rnai and epigenetics source book

RNAi AND EPIGENETICS

SOURCEBOOK

RNAi AN

D EPIGEN

ETICS SOURCEBO

OK

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by life technologies

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Quick start guide

1. Decideoneffectormolecule:siRNAorvector—Chapter1

2. Find your gene online: www.invitrogen.com/findyourgene

(shownatright)

3. Choose delivery vehicle: transfection reagent, mechanical

method,orviraldelivery—Chapter3

4. Decideoncontrols—Chapter2

5. Validatetomeasurelossoffunction—Chapter11

Figure 1. RNAi workflow diagram. RNAiexperimentworkflowfollowingsiRNAdesignandsynthesis.

Steps of an siRNA experiment

Haveonhand:

→ Transfection/electroporationagentandprotocol

→ AssaystoassessknockdownandotherRNAieffect(s)

→ PositiveandnegativecontrolsiRNAs

→ TwoormoresiRNAstogeneofinterest

Find and order siRNAs at:www.invitrogen.com/RNAi

Monitor siRNA-induced knockdown to:• Validate the siRNA• Monitor transfection efficiency

Observe/measure phenotypic change

Plate cells and transfect siRNAs

1

2

Prep RNA3

4 4

10

100 5 15

Cycle

Control siRNA

Survivin siRNA

20 25 30 35 40

1

0.1

0.010

20

40

60

80

100

NEK

4-1

NEK

4-2

NEK

4-3

TGFB

R2-

1TG

FBR

2-2

TGFB

R2-

3

STK

6-1

STK

6-2

STK

6-3

RP

S6K

A4-

1R

PS

6KA

4-2

RP

S6K

A4-

3

MA

P2K

1-1

MA

P2K

1-2

MA

P2K

1-3

MA

P2K

4-1

MA

P2K

4-2

MA

P2K

4-3

WEE

1-1

WEE

1-2

WEE

1-3

PK

428-

1P

K42

8-2

PK

428-

3

STK

12-1

STK

12-2

STK

12-3

PC

NA

gold

% R

NA

rem

aini

ng

30

t5'3'

3'5'

Rn

Glossary of common RNAi terms

RNAiRibonucleicacidinterference(firstusedbyA.FireandC.Melloetal.,1998).

siRNAShortinterferingRNA.siRNAsare21–25bpdsRNAwithdinucleotide3’overhangsthatareprocessedfromlongerdsRNAbyDicerintheRNAinter-

ferencepathway.IntroductionofsyntheticsiRNAscaninduceRNAinterferenceinmammaliancells.siRNAscanalsooriginatefromendogenous

precursors.

shRNAShorthairpinRNA;also short interferinghairpin. shRNAsareused invector-basedapproaches for supplyingsiRNA tocells toproducestable

genesilencing.AstrongPolIIItypepromoterisusedtodrivetranscriptionoftargetasequencedesignedtoformhairpinsandloopsofvariable

length,whichareprocessedbycellularsiRNAmachinery.OnceinthecelltheshRNAcandecreasetheexpressionofagenewithcomplementary

sequencesbyRNAi.

miR RNAiVectorsthatexpressmicroRNAsforRNAi.miRNAsare19–23ntsingle-strandedRNAs,originatingfromsingle-strandedprecursortranscriptsthat

arecharacterizedbyimperfectlybase-pairedhairpins.miRNAsfunctioninasilencingcomplexthatissimilar,ifnotidentical,toRISC.

Chemically modified siRNAsiRNAmoleculeswhichhavechemicalmodifications.

RISCRNA-induced silencing complex (RISC). A nuclease complex, composed of proteins and siRNA, that targets and cleaves endogenous mRNAs

complementarytothesiRNAwithintheRISCcomplex.

Off-target effectsEffectsthatoccurwhenoneorafewnontargetgenesnotspecificallytargetedshowlossofgenefunctionfollowingtheintroductionofansiRNA

ord-siRNApool.TheeffectmaybemediatedbythesensestrandofansiRNA,whichmayinitiatealoss-of-functionresponsefromanunrelated

gene.Off-targeteffectscanalsooccurasasecondaryeffectoftheantisensestrandofaspecificsiRNAifithassufficienthomologytoknockdown

theexpressionofanontargetgene.

i

Contents

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SECTION I—RNA INTERFERENCE

CHAPTER 1

Introduction to RNAi .................................................................... 2

MakeyourRNAinterferenceexperimentssimple,stress-free,andsuccessful.................................... 2

AbriefhistoryofRNAi ................................................................... 2

HowRNAiworks ...................................................................... 2

EighttipsforasuccessfulsiRNAexperiment...................................................... 3

Considerations ....................................................................... 3

InterviewwithGregoryHannon ............................................................. 4

CHAPTER 2

Controls for RNAi experiments............................................................. 7

Transfectioncontrols................................................................... 7

Negativecontrols ..................................................................... 8

Positivecontrols...................................................................... 8

Downstreamcontrols................................................................... 9

Interferoncontrols..................................................................... 9

UsemultiplesiRNAsequencespertargettoverifyresults ............................................... 9

TitratesiRNA........................................................................ 9

Rescueexperiments .................................................................... 9

CHAPTER 3

Delivering RNAi to cells—transfection and viral delivery ............................................ 10

Methodstoachievehightransfectionefficiency................................................... 10

Importanceofminimizingtransfection-mediatedcytotoxicity ........................................... 10

Significanceofreducingoff-targeteffects....................................................... 11

Cellhealth ........................................................................ 14

Cultureconditions .................................................................... 14

Passagenumber ..................................................................... 14

siRNAquality ....................................................................... 15

siRNAquantity ...................................................................... 15

Choiceoftransfectionagent.............................................................. 15

Volumeoftransfectionagent............................................................. 15

Exposuretotransfectionagent/siRNAcomplexes.................................................. 15

Presenceofseruminthemediumduringtransfection................................................ 16

Optimizingtransfectionexperiments ......................................................... 16

ContentsX

Contents

ii

Section IIIRNAi and Epigenetics Sourcebook

CHAPTER 4

Vector-based RNAi technologies ........................................................... 17

IntroductiontoadenoviralandlentiviralRNAivectorsfordelivery ......................................... 17

LentiviralandadenoviralRNAivectors—chooseanycelltypeforRNAi...................................... 18

CHAPTER 5

In vivo RNAi........................................................................ 19

RNAimoleculesforin vivo applications........................................................ 19

ChoosingRNAieffectormolecules ........................................................... 19

ChoosingRNAipurity.................................................................. 20

Trackingdeliveredduplexes.............................................................. 21

In vivoRNAiprotocols..................................................................22

MeasuringRNAconcentration .............................................................23

Harvestingtissue—RNAextractionfromtissue .................................................... 24

Sectioningtissue.................................................................... 24

Proteinextractionfromtissues .............................................................25

Frequentlyaskedquestionsaboutin vivoRNAi.................................................... 27

CHAPTER 6

siRNA screening...................................................................... 29

SelectingansiRNAlibrary................................................................ 29

ScreeningwithsiRNAlibraries ............................................................. 31

siRNAlibraryscreeningworkflow(5steps)...................................................... 32

Toxicity ..........................................................................34

Secondaryscreening:confirmationofcandidates.................................................. 35

SECTION II—PRODUCTS FOR RNA INTERFERENCE

CHAPTER 7

siRNA technologies ....................................................................40

Ambion®Silencer®SelectsiRNAandStealthRNAi™siRNA..............................................40

siRNAselectionguide ..................................................................48

Silencer®SelectsiRNAlibraries............................................................. 50

Silencer®Pre-designedandValidatedsiRNAs..................................................... 52

CHAPTER 8

Vector-based RNAi technologies ........................................................... 53

Howvector-mediatedRNAiworks ........................................................... 56

BLOCK-iT™PolIImiRRNAiexpressionvectors—versatilePolIIvector-basedRNAitechnology.......................... 57

iii

Contents

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CombineBLOCK-iT™vectorkitswithlentivirusforstable,long-termexpression................................. 58

Gateway®compatibilityforexpandedexperimentaloptions ............................................ 62

BLOCK-iT™miRRNAiSelect ...............................................................64

GuaranteedresultswithBLOCK-iT™miRRNAiSelect4Sets ............................................. 65

BLOCK-iT™RNAiEntryVectorKits...........................................................66

LentiviralandadenoviralRNAivectors.........................................................68

PowerfulshRNAdeliverywithBLOCK-iT™viralvectors ................................................ 69

BLOCK-iT™LentiviralRNAiSystem ........................................................... 70

BLOCK-iT™AdenoviralRNAiExpressionSystem.................................................... 71

CHAPTER 9

RNAi delivery....................................................................... 72

IntroductiontoRNAidelivery.............................................................. 72

Lipofectamine®RNAiMAXTransfectionReagent—unmatchedgenesilencingwithreducedcytotoxicityforsiRNAexperiments....... 73

Lipofectamine®2000andLTXTransfectionReagents—idealfordeliveryofshRNAandmiRRNAivectors................... 75

Oligofectamine™TransfectionReagent—potentinternalizationofRNAoligos.................................. 76

TheNeon™TransfectionSystem ............................................................ 76

CHAPTER 10

RNA interference controls............................................................... 79

ControlsforRNAiexperiments............................................................. 79

BLOCK-iT™AlexaFluor®RedFluorescentControlandgreenBLOCK-iT™FluorescentOligo ............................80

BLOCK-iT™TransfectionKit—simpleoptimizationofLipofectamine®2000transfectionconditions....................... 81

Silencer®SelectpositiveandnegativecontrolsiRNAs................................................ 82

StealthRNAi™siRNAnegativecontrols—idealnegativecontrolsforeveryexperiment.............................. 83

StealthRNAi™siRNAreportercontrols .........................................................84

StealthRNAi™siRNApositivecontrols .........................................................84

BLOCK-iT™TransfectionOptimizationKit(Human)..................................................84

OptimizeRNAiexperimentswiththeBLOCK-iT™FluorescentOligo......................................... 85

PositivecontrolsforBLOCK-iT™PolIImiRRNAivectors................................................ 85

Silencer®negativecontrolsiRNAs............................................................86

Silencer®positivecontrolsiRNAs ............................................................ 87

CHAPTER 11

Measuring knockdown.................................................................88

FunctionalvalidationfollowingRNAiknockdown..................................................88

TaqMan®GeneExpressionAssays...........................................................88

CustomTaqMan®GeneExpressionAssays....................................................... 89

CustomTaqMan®ProbesandPrimers ......................................................... 89

qRT-PCRdirectlyfromcells...............................................................90

Proteinseparationandwesternblotting....................................................... 91

ContentsX

Contents

iv


Antibodiesandimmunodetection ........................................................... 91

Nucleicacidpurificationandquantification...................................................... 91

Geneexpressionmicroarrayanalysis.......................................................... 91

CHAPTER 12

RNAi services ....................................................................... 92

RNAiservices....................................................................... 92

RNAidesignservices.................................................................. 93

StealthRNAi™,Silencer®Select,andSilencer®siRNAcustomsynthesis ........................................ 93

Vector-basedRNAiservices ............................................................... 93

Deliveryoptimizationservices .............................................................94

Viralproduction..................................................................... 95

RNAifunctionalvalidationservices..........................................................96

Phenotypicassaydevelopmentandhigh-throughputscreeningservices..................................... 97

RNAicustomcollaborativeresearch.......................................................... 97

CHAPTER 13

Overview of miRNAs...................................................................98

WhichmiRNAprofilingorvalidationsystemshouldIuse? ..............................................99

Genome-widesmallRNAdiscoveryandprofiling..................................................100

MicroRNA:anewfrontierinbiology.........................................................100

FundamentalquestionsinmicroRNAresearch ...................................................100

CapturethesequenceandexpressionlevelofeverysmallRNAinyoursample................................. 101

Genome-widesmallRNAdiscoveryandprofilingworkflow............................................ 101

RobustsmallRNAdiscoveryandprofilingusingtheleadingnext-generationsequencingplatform......................102

ProfilemicroRNAexpressioninasingledayusinggoldstandardTaqMan®assaytechnology.........................103

DetectandquantifyspecificmicroRNAs......................................................106

AnalyzemicroRNAfunction.............................................................108

SamplepreparationtailoredformicroRNAexperiments ...............................................112

CHAPTER 14

Optimized miRNA profiling...............................................................115

NCode™platform—foroptimizedmiRNAprofiling ..................................................115

EfficientisolationoftotalRNA(includingmiRNA)usingTRIzol®Reagent..................................... 116

ReliablerecoveryoftotalRNA(includingmiRNA)fromFFPEsampleswiththeRecoverAll™TotalNucleicAcidIsolationKit ......... 116

SimplifiedmiRNAfluorescentlabeling ........................................................ 118

UltrasensitivemiRNAamplification..........................................................119

ComprehensivemiRNAexpressionprofiling....................................................120

NCode™Non-codingRNAArrays........................................................... 124

Genome-wideanalysisoflongnon-codingRNA(ncRNA)expressioninsolidtumorsusingtheNCode™Non-codingRNAMicroarrayplatform ............................................ 126

v

Contents

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SECTION III—miRNA PROFILING

CHAPTER 15

Introduction to DNA methylation..........................................................134

OverviewofDNAmethylation ............................................................134

DNAmethylationanalysis...............................................................134

MethylatedDNAenrichment .............................................................134

EnrichmentofdifferentiallymethylatedregionswithMethylMiner™fractionationanddeepsequencingwiththeSOLiD™System....136

Locus-specificDNAmethylationanalysis...................................................... 142

Genome-widemethylation .............................................................. 142

PureLink™reagents—reliableisolationofgenomicDNA.............................................. 143

MethylCode™bisulfitetreatment—fastandcompleteDNAconversion.....................................144

Detectionofbisulfite-modifiedDNA.........................................................145

High-fidelityreagentsforamplifyingbisulfite-treatedDNAandmethylation-specificPCR(MSP)* .......................145

Simplifyyourpreparationforbisulfitegenomicsequencing............................................146

Top-ratedsequencingvectors ............................................................146

SECTION IV—HISTONE MODIFICATIONS AND CHROMATIN REMODELING

CHAPTER 16

Histone modifications and chromatin remodeling ................................................150

Investigatinghistonemodificationsandchromatinremodeling......................................... 151

MAGnify™ChIP.................................................................... 151

MAGnify™ChromatinImmunoprecipitationSystem ................................................ 152

Antibodiesqualifiedforchromatinimmunoprecipitation .............................................154

DynaMag®-PCR....................................................................154

QuantitativePCR(qPCR)ofChIP-readyDNA..................................................... 155

ContentsX

Contents

Section I—RNA Interference

Chapter 1—Introduction to RNAi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Chapter 2—Controls for RNAi experiments . . . . . . . . . . . . . . . . . . . . . . . 7

Chapter 3—Delivering RNAi to cells—transfection and viral delivery . . . . . .10

Chapter 4—Vector-based RNAi technologies . . . . . . . . . . . . . . . . . . . . . 17

Chapter 5—In vivo RNAi. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Chapter 6—siRNA screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

2Important Licensing Information:TheseproductsmaybecoveredbyoneormoreLimitedUseLabelLicenses(See InvitrogenCatalogorourwebsite,www.invitrogen.com).Byuseoftheseproductsyou accept the terms and conditions of all applicable Limited Use Label Licenses. All products are for research use only. CAUTION: Not intended for human or animal diagnostic or therapeutic uses.

Section I RNA Interference

CHAPTER 1

Introduction to RNAi

Make your RNA interference experiments simple, stress-free, and successful

RNAinterference(RNAi)isoneofthemostimportanttechnologicalbreakthroughsinmodernbiology,allowingustodirectlyobservetheeffects

ofthelossoffunctionofspecificgenesinmammaliansystems.Onceviewedasatechniqueusedonlybyselectlaboratories,RNAiisnowconsid-

eredessentialforstudyinggenefunction.Ithasbecomeaprominenttoolforproteinknockdownstudies,phenotypeanalysis,functionrecovery,

pathwayanalysis,in vivoknockdown,anddrugtargetdiscovery.

A brief history of RNAi

Inthe early1990s,scientistsfirstobservedthatRNAinhibitedproteinexpressioninplantsandfungi.In1998,AndrewFireandCraigMello,working

withCaenorhabditis elegans,discoveredthatdouble-strandedRNA(dsRNA)wasthesourceoftheinhibition,andtheycalledthisphenomenon

RNAinterference(RNAi).WhilestudiesinC. eleganswereencouraging,theuseofRNAiwaslimitedtolowerorganismsbecausedeliveringlong

dsRNAforRNAiwasnonspecificallyinhibitoryinmammaliancells.In2001,shorterRNAs(siRNA)wereshowntodirectlytriggerRNAiinmammalian

cells,withoutevokingthenonspecificeffectsobservedwithlongerdsRNAs.In2006,just8yearsafterthediscoveryofsiRNA,theNobelPrizein

PhysiologyorMedicinewasawardedtoFireandMellofortheirdiscovery,underscoringtheimportanceofRNAiasaninvestigativetool.

How RNAi works

The molecules that mediate RNAi are short

dsRNA oligonucleotides, 21 nucleotides in

length, which are processed internally by

an enzyme called Dicer. The Dicer cleav-

age products were first referred to as short

interfering RNA, now known as siRNA. RNAi

technology takes advantage of the cell’s

natural machinery to effectively knock

downexpressionofagenewithtransfected

siRNA.ThereareseveralwaystoinduceRNAi:

synthetic molecules, RNAi vectors, and in

vitro dicing (Figure 1.1). In mammalian cells,

short pieces of dsRNA—short interfering

RNA—initiate thespecificdegradationofa

targeted cellular mRNA. In this process, the

antisense strand of siRNA becomes part of

a multiprotein complex, or RNA-induced

silencingcomplex(RISC),whichthenidenti-

fiesthecorrespondingmRNAandcleaves it

ataspecificsite.Next,thiscleavedmessage

istargetedfordegradation,whichultimately

resultsinthelossofproteinexpression.

Silencer® Select siRNA

Stealth™ RNAi™ siRNA

shRNA expression

siRNA in mammalian cell

Target mRNA

Cellular message is destroyed

miRNA expression Dicer processing of long dsRNA

(pool of 21–23 nt siRNAs)

Target cleavage

RISC loading siRNA unwindingTarget recognition

Dicer

Synthetic molecules for RNAi Vector-based expressed RNAi In vitro synthesized RNAi

Figure 1.1. Methods of RNAi knockdown in mammalian cells.

RNA

Inte

rfere

nce

I

3

Chapter 1Introduction to RNAi

www.invitrogen.com

Eight tips for a successful siRNA experiment

1. Go to www.invitrogen.com/rnai and utilize the best-in-class design algorithms to design your siRNAs. DonotattempttodesignsiRNAs

onyourown.

2. Avoid RNases! Trace amounts of ribonucleases can sabotage siRNA experiments. Since RNases are present throughout the laboratory

environmentonyourskin,intheair,onanythingtouchedbybarehandsoronanythingleftopentotheair,itisimportanttotakestepsto

preventandeliminateRNasecontamination.AmbionoffersacompletelineofproductsdesignedtodetectandeliminateRNases.

3. Maintain healthy cell cultures and strict protocols for good transfection reproducibility. Ingeneral,healthycellsaretransfectedathigher

efficiencythanpoorlymaintainedcells.Routinelysubculturingcellsatalowpassagenumberensuresthattherewillbeminimalinstabilityin

continuouscelllinesfromoneexperimenttothenext.Whenperformingoptimizationexperimentswerecommendtransfectingcellswithin

50passages,sincetransfectionefficiencydropsovertime.

4. Avoid antibiotic use. Avoid theuseofantibioticsduringplatingandupto72hoursafter transfection.Antibioticshavebeenshownto

accumulate to toxic levels in permeabilized cells. Additionally, some cells and transfection reagents require serum-free conditions for

optimalsiRNAdelivery.Wesuggestyouperformapilottransfectionexperimentinbothnormalgrowthmediaandserum-freemediato

determinethebestconditionforeachtransfection.

5. Transfect siRNAs using optimized reagents. UseanoptimizedsiRNAtransfectionreagentandprotocolforyourcelltype.Thechoiceof

transfectionreagentiscriticalforsuccessinsiRNAexperiments.ItisessentialtousetransfectionreagentsformulatedtodeliversmallRNAs

(mostcommerciallyavailabletransfectionreagentsweredesignedforlargeplasmidDNA,notsmallRNAmolecules).Also,somereagents

havebeendevelopedforthetransfectionofspecificcelllineswhileothershavebroaderspecificity.

NOTE: Lipofectamine® RNAiMAX (Cat. No. 137780-75) is our best-performing transfection reagent for siRNA. See page 73 for more information.

6. Use an appropriate positive control to optimize transfection and assay conditions. Housekeepinggenesaresuitablepositivecontrols

formostcelltypes.Tooptimizeconditions,transfecttargetcellswithseveralconcentrationsofansiRNAspecifictoyourchosenpositive

controlandtoyourexperimentaltargetsiRNA.MeasurethereductioninthecontrolproteinormRNAlevelcomparedtountransfectedcells

48hoursaftertransfection.ToomuchsiRNAcanleadtocelltoxicityanddeath.Formaximumconvenience,Invitrogenofferspositivecontrol

siRNAsagainstavarietyofgenetargets.

7. Use a negative control siRNA to distinguish nonspecific effects. Negative controls should be designed by scrambling the nucleotide

sequenceofthemostactivesiRNA.However,besuretoperformahomologysearchtoensurethatyournegativecontrolsequencelacks

homologytothegenomeoftheorganismbeingstudied.

8. Use labeled siRNAs for protocol optimization. Fluorescently labeled siRNA can be used to analyze siRNA stability and transfection

efficiency.LabeledsiRNAisalsousefultostudysiRNAsubcellularlocalizationandindoublelabelexperiments(withalabeledantibody)to

visualizecellsthatreceivesiRNAduringtransfectionandtocorrelatetransfectionwithdown-regulationofthetargetprotein.

Considerations

siRNA vs. vector approachesBothsiRNAandvector-basedRNAicanbeextremely

effectiveatproducinglossoffunctionphenotypes.

Ingeneral,mostresearcherschoosesiRNAbecause

theycanstartquicklyandtherearenospecialprepa-

rations needed other than basic cell culture tech-

niques.However,thereareanumberofreasonswhy

aresearchermightchooseeithersiRNAoravector-

basedRNAi.Table1.1containscriteriathatwillhelp

youmakethedecision.

Table 1.1. Synthetic siRNA vs. vector-expressed siRNA.

siRNA RNAi Vectors miR RNAi & shRNA

Long-termstableknockdown •

Inducibleknockdown •

Deliverytohard-to-transfectcells •

Leasthands-ontime •

Mostimmediateeffect •

Higherpotencylikely •

Designmoreefficient •

Introduction to RNAi1



CHOOSINGsiRNA FOR TARGET OF INTEREST—IMPROVED SPECIFICITYWhenchoosingsiRNAforatargetofinterestitisveryimportanttouseastate-of-the-artalgorithmforchoosingyoursiRNA.Forexample,

Silencer®SelectsiRNA’sfive-stepbioinformaticfilteringprocessallowsforeliminationofsiRNAsthatarepredictedtoelicitoff-targeteffects.

www.invitrogen.com/silencerselect

WithStealthSelectRNAi™siRNA,theworkofdesigninghighlyeffectivesequenceshasbeendoneforyou.Thedevelopmentofthese

moleculesbeginswithidentifyingallhuman,rat,andmousetranscriptsintheUnigenedatabase.Infourstepsweselecthighlyeffective

in silico-designedsequences.www.invitrogen.com/stealthrnai

Seepage40forSilencer®SelectsiRNAandpage44forStealthSelectRNAi™siRNA.

Interview with Gregory Hannon

Q. Research into RNA interference (RNAi) and its emerging use as a tool to explore gene function has taken the research community by storm. Can you tell us how you first became interested in RNAi?Dr. Hannon:It’ssortofinteresting,actually.IwasataPewScholarsMeeting;itwasmyfirstyear.

CraigMellowasalsoaPewScholar,andhegaveasmall“chalktalk”atthemeeting—althoughwewereinthebowelsofMexico

somewhere,sotherewasnochalk.Hepresentedthisreally interestingphenomenon. Itwaseitherjustbeforeorjustafterthefirst

RNAipaperwaspublished.Itreallyexcitedmeandwechattedacoupletimesaboutitatthemeeting.Butwedidn’treallydoanything

aboutit,notbeingC. elegansbiologists.Histheoryjustsortofpercolatedforayear.

Thenthenextyear,againatthePewScholarsMeeting,RichCarthewshowedthatRNAiworkedinDrosophila.Hewasdoingthis

byembryoinjection.Andthat’swhatreallypulledmein.HerewassomethingthatwasnotjustabiologicaloddityofC. elegans (Iwas

unawareoftheplantworkatthattimesoIhadn’tthoughtaboutitverydeeply).Thenotionthatthisphenomenonwasgoingtobe

universalreallycaptivatedme.ThiswaspartlybecauseIwasspendingalotofefforttryingtodoforwardgeneticsinculturedmamma-

liancellsusingthingslikeretrovirallibraries.IsawtheRNAiphenomenonas,onedaylongdowntheroad,somethingthatcouldcom-

plementoverexpressionapproaches,andthatwouldgiveustheloss-of-functiontoolthatmammaliansystemshavealwayslacked.

Q. Wow, “…one day long down the road…”, I bet you were surprised at how quickly this field has progressed.Dr. Hannon:Well,that’swhatgotusstartedinRNAi.WestartedthinkingaboutthecellculturemodelsfromDrosophila.Ourinitialgoal

hadbeentotrytouseS2cellsasamodelforstudyinggenefunction.AndsoIactuallycalledthelabfromthatPewMeetingandsaid,

“…Youknowwhat?GetoutthoseDrosophilacellsandseeiftheydoRNAi…”Andtheydid.

Q. So how did your interest in using RNAi to study gene function morph into your work focusing on the mechanism of RNAi?Dr. Hannon:WegothookedonthistechniqueinpartbecauseIhaveabackgroundinRNAprocessing—Iworkedontrans-splicing

withTimNilsenasagraduatestudent—sotherewasthepossibilitythatwewouldhavejustrunwiththewholenotionofdoinggene

functioninS2cells.Asitturnsout,formostofthethingsthatwewereinterestedin—cellcyclecontrol,andsuch—S2cellsareater-

riblesystem.Butwesaid,okay,sincenobodyunderstandsmuchabouttheRNAimechanismyet,let’splayforalittlewhileandseeif

wecangenerateanin vitrosystemfromculturedcellsthatwecanusetotrytofigureouthowthisallworks.Andwesortofgotsucked

downthemechanismpathfromthere.

Q. Your lab, Mello’s lab, and others published research indicating that Dicer is the nuclease that digests long dsRNA into siRNAs which in turn mediate RNAi. Can you describe some of the experiments that led you to this conclusion?Dr. Hannon:Wehadapproachedthispurelyfromabiochemicalstandpoint,wherewehadtakena“candidategeneapproach”.Wetagged

alloftheseproteins,didIPs(immunoprecipitations),andlookedforactivity.ItledtoabeautifulbiochemicalcorrelationbetweenDiceractiv-

ityandsiRNA.Inotherwords,wehadanenzymethatdidbasicallywhatitwassupposedtodo.Butanytimeyouhaveabiochemicalresult,

youhavetobesomewhatconcernedaboutwhetherornotthestoryyou’vederivedbasedonin vitroexperimentshasanybasisinreality.

Toreallyknowthatsomethingisinvolvedinanykindofspecificpathwayyouneedgenetics.Ultimatelytheproofisalwaysinthe

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Chapter 1Introduction to RNAi

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genetics.Andwehaddoneareallycrappyexperiment,whichwastouseRNAitoknockoutDicer—that’ssortofbecomederigueurinthe

field—soIdon’tknowifIshouldstillbeembarrassedaboutornot.ButourprimarymotivationwasjusttoverifythehypothesisthatDicer

wastheinitiatingenzyme.

Q. Now we know that Dicer is also important in a gene regulatory pathway involving short temporal RNA (stRNA) and your lab’s work helped to demonstrate that. How did you tie Dicer to the stRNA pathway?Dr. Hannon:ShortlyafterEmilyleft,Plasterk’slabfinallyfoundaC. elegansDicermutant.Itwasinitiallysomewhatworryingthatthere

weren’tsomaticRNAidefects.Ronaldtriedtobereassuringandtalkedaboutmaternaleffects.Butultimatelywhentheylookedat

germlinetransgenes,silencingofgermlinetransgeneswasdefective.Andthiswassortofaniceconfirmation.Ithinkatleastnow

there’snodoubtthatDicerreallyistheinitiatingenzymefortheprocess.

Q. What do you think will be the single greatest outcome of research into RNA silencing?Dr. Hannon:It’sreallyanimpossiblequestiontoanswer.Oneofthethingsthatmakesthisfieldveryhotatthemomentisthatina

wayit’sallthingstoallpeople.Youhaveveryinterestingbiology.Forexample,scientistsarestudyingtransposonsilencing,theevolu-

tionoftherelationshipbetweenrepetitiveandmobilegeneticelements,DNAparasitesandtheirhosts,andtheinteractionbetween

virusesandtheirhosts.ThatgroupisinterestedbecauseRNAiisinvolved,atleastinplants.Andthereisalsoadefinitiverelationshipin

C. elegans.RNAiseemstobeinvolvedinsomehowcontrollingthesekindsofnucleicacidparasites.Buttherearealsopeoplelooking

atthesemicroRNAs,atleastforendogenousgeneregulation,andsaying,look,here’sawholenewregulatoryparadigmwherethere

arehundredsof,whatyoumightcallorphanhairpins,runningaroundoutthere.Wedon’tknowwhattheydo,wedon’tknowwho

theyregulate,wedon’tknowhowprevalentthisis,howgeneralitis,orevenreallyatwhatregulatorylevelsthesedifferentthingsact.

Weknowabouttwothatactatthelevelofproteinsynthesis,butthere’snothingtosaythatothersdon’tactatthelevelofmessage

stability,orevenatthelevelofdirectingthemodificationofchromosomestructure.Herewemayfindthatthisissomethingthatis

asimportantasthediscoveryofenhancersequences,intermsofcontrollinggeneexpressionitself.

You’vealsogotagroupofpeoplewhoareinterestedinthebasicmechanicsofRNAiandwhatitmeans.Everybodyisintrigued

aboutamechanismwhereawormcaneatapieceofRNAandknockoutageneinitsprogeny.There’ssomethingintrinsicallyappeal-

ingaboutthat—understandingthemechanismofthatbitofbiology.Andthere’sawholegroupofpeoplewhowanttounderstand

thebiologicalramificationsofthesystem—thatitmayregulatedevelopmentinplants,maybestemcellidentity,ormaintenanceof

stemcellcharacterinbothplantsandanimals.

Andthen,amuchbroadergroupofpeople,whodon’treallycarethatmuchaboutmechanism,arejustinterestedinharnessingthis

phenomenonasatool.So,it’sreallyimpossibletopredictthesinglegreatestoutcomeofthisresearch.AndIthinkifyouaskthatquestionof

tendifferentpeople,you’dgettendifferentanswers.ThereasonIcan’tgiveyoujustoneisbecausewe’reinterestedinallofthem.

Q. Your lab and other labs have developed expression vectors that express siRNA long term. Do you see the use of siRNA expression vectors as a replacement to transfection of siRNAs for inducing RNAi, or are these techniques complementary?Dr. Hannon:Oh,Ithinkthey’recomplementary.Verymuchso.It’sstillearlyintermsoftryingtounderstandthepowerofeachof

thesetechnologies.Whatseemstobetrue,atleastthisearlyon,isthatsiRNAscangetintocellsatverylowconcentrationstoprovoke

averygoodeffect.Ithinkthejuryisstilloutonwhetherit’seasiertogetaneffectwithansiRNAonasortofpercellorwholepopula-

tionbasis.Wedon’treallyhavethatmuchinformationonit.AndIsuspecteventuallythesethingswillrunevenbecauseofadvances

indifferenttransfectiontechnologies.Butintermsofeaseofuse,nothingbeatstypinginasequence,havingacoupleoligosshow

up,andthendumpingthemontocells,right?Ifyou’relookingforaquickanswer,andyouonlyhaveoneortwogenesthatyouwant

tolookat,nothingisgoingtobeattheideathatyoucanchemicallysynthesizethesethings,justintermsofease.

Q. What are some of the advantages of expression vectors over chemically synthesized siRNAs?Dr. Hannon:Theexpressionconstructsaregoingtobepowerfulforadifferentreason.Andmaybemoreappropriateforspecific

sortsofexperimentsthatinvolvemuchmorelongtermanalysisofphenotype,orbiochemicalstudiesthatinvolvelargercellnum-

bersthatmightbemoredifficultormoreexpensivetodobytransfectingeachcellthatyouwanttoanalyze.

Q. Cell logistic problems, right?Dr. Hannon:Right.Therearealotofphenotypesthatyouwanttolookatoverlongtimescalesorinmosaicanimals.That’swherethe

powerofthesekindsofexpressionconstructsaregoingtobe.Now,anotheradvantageoftheexpressionconstructsisthefact

Introduction to RNAi1



thattheyarepropagatable—youmakeoneandvalidateitandyouhaveitforever.Youneverhavetoremakeit.Youneverhavetoreorder

it—itsitsinthefreezerandyoucantrotitoutanduseitanytimeyouwant.Wearefindingthatyoucanmarrythesesortsofmodularcas-

setteswithprettymuchanygenetransfertechnologythatyouwanttotalkabout—viruses,etc.Theywillbeusefulwithmodelsystems

liketissueslices,whereitmightbemoredifficulttogetsiRNAs[insidecells]ingoodnumbers.

Q. I’d like to ask you about your laboratory. How many people do you have working with you currently, and how are they split between postdocs, grad students, etc.?Dr. Hannon:Weareataround16atthemoment;6graduatestudents,acoupleofvisitors,about4or5postdocs,me,afewtechnicians,

andaresearchassociatewhoissemi-independentwho’salsoworkingwithme.

Q. We know that you’re the founder of the biotech company, Genetica. And you’re obviously busy as a professor at the Cold Spring Harbor labs, too. Do you still find time to work at the bench? And, if so, do you think that’s unusual for someone in your position?Dr. Hannon:Yeah,oh,definitely.Itrytoworkatthebencheveryday.I’mnotsurethatIdoanythinguseful,butItrytowork.Ithinkthat

ColdSpringHarborhas,notnecessarilyatradition,butalotofthefacultyheredoworkatthebench.And,ifyouthinkaboutit,it’ssort

ofwhatgotusinvolvedinthiswholeRNAiworkinthebeginning.Ifindthatitkeepsmeengagedmuchmoreinthedaytodayactivities

ofthelab.Italsokeepsmegroundedintherealityofdoingexperimentsandmakesmemuchmoreunderstandingaboutstudents’liga-

tionsfailingoccasionally,becauseminefailrightalongsidetheirs.AndIthinkthattheworkmovesfasterbecauseI’mthere.I’mavailable.

Wetalkaboutthingsmore—everythingfromthebiologyofRNAitothenittygrittydetailsoflabwork,like,“Gee,thisisn’tworking,”and

“MaybeI’veseenthatbefore…”Thathelpsustroubleshootandmovethingsalongalittlebitmorequickly.Ilikebeinginthelab,andIlike

interactingwiththepeopleinmylab.

Q. What’s next for Gregory Hannon?Dr. Hannon:I’mgoingtogodominiprepsforoneofmystudents,that’swhat’snext.

About Dr. Hannon

Gregory J. Hannon received his PhD from Case Western Reserve University in 1992 and was a 1997 Pew Scholar. He is currently an Associate Professor

at the Cold Spring Harbor Laboratory, where his research is focused in part on determining the mechanism of RNAi, as well as using RNAi as a tool

in the study of cancer development and investigating the potential of siRNAs as cancer therapeutic agents.

Dr. Hannon and his colleagues have been at the forefront of many of the important discoveries in the RNAi area. In 2000 they identified the nuclease

activity responsible for dsRNA-guided mRNA degradation, now known as the RNA-induced silencing complex (RISC). They identified one of the protein

components of RISC, Argonaute2, as well as the enzyme (“Dicer”) that begins the RNAi process by cutting long dsRNAs into siRNAs. Most recently they have

shown the effectiveness of short hairpin RNAs (shRNAs) at gene silencing, providing a longer-lasting alternative to siRNAs for much-needed functional

studies.

Dr. Hannon is one of the founders of Genetica, Inc., a biotechnology company using RNAi and other tools to link genetic data with biologic

function via genetic manipulation of mammalian cells. Such genetic manipulations include RNAi-mediated stable silencing of gene expression for

the elucidation of disease pathways such as cancer development and subsequent drug target validation.

References1. PaddisonPJ,HannonGJ(2002)Cancer Cell2(1):17–23.

2. HannonGJ(2002)Nature418(6894):244–251.

3. PaddisonPJ,CaudyAA,BernsteinE,HannonGJ,ConklinDS(2002)Genes Dev16(8):948–958.

4. PaddisonPJ,CaudyAA,HannonGJ(2002)Proc Natl Acad Sci U S A99(3):1443–1448.

5. BernsteinE,DenliAM,HannonGJ(2001)RNA7(11):1509–1521.

6. HammondSM,BoettcherS,CaudyAA,KobayashiR,HannonGJ(2001)Science293(5532):1146–1150.

7. HammondSM,CaudyAA,HannonGJ(2001)Nat Rev Genet2(2):110–119.

8. BernsteinE,CaudyAA,HammondSM,HannonGJ(2001)Nature409(6818):363–366.

9. HammondSM,BernsteinE,BeachD,HannonGJ(2000)Nature404(6775):293–296.

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Chapter 2Controls for RNAi experiments

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CHAPTER 2

Controls for RNAi experimentsPropercontrolsareessentialtoensuresuccessineveryRNAiexperiment.Thenumberandtypesofcontrolschosendependsupontheultimate

researchgoal(Table2.1).WehavesimplifiedcontrolreactionsbyprovidingaselectionofRNAitechnologiesdesignedtoassistresearchers in

identifyingandvalidatingdrugtargets,generatingpublishabledata,andsubmittinggrants.OurRNAicontrolkitsallowyouto:

→ DeterminewhichRNAimoleculesdeliverthebestknockdownresults

→ Achievegreaterknockdownbyoptimizingtransfectionprotocols

→ Savetimebyconfirmingcellviabilityearlyinanexperiment

→ ProceedwithconfidencebycomparingtargetedRNAireagentstoasetofreagentsoptimizedforinhibitionofp53inhumancells

Table 2.1. RNAi interference controls.

Type of control Recommended use Products

Transfectioncontrol Calculateandmonitortransfectionefficiencywithfluorescence Seepage80

NegativecontrolsNonspecificorscrambledcontrolsusedtomeasureknockdownlevelsvs.background

Seepage82,83,and86

PositivecontrolsRNAireagentsknowntoachievehighlevelsofknockdownusedtomeasuredeliveryandoptimizeexperimentalconditions

Seepage82,84,and87

Untransfectedcontrol Measurenormalgeneexpressionlevelandphenotype

MultipleRNAisequencestothesametarget

Usetoverifyphenotypicchange,controlforoff-targeteffectsforgeneratingpublicationqualityresults

TitrationofRNAi Usethelowesteffectiveleveltoavoidalteringthecellsnormalprocesses

RescueexperimentsTurnoffinducibleRNAiorintroduceaplasmidexpressingthetargetmRNAthattheRNAisequencewillnotaffect

BLOCK-iT™PolIImiRRNAiorBLOCK-iT™shRNAvectorswithinduciblepromoters(CMV/TOandH1/TOrespectively)(seepage57)

Transfection controls

Withoverexpressionexperiments,evenlowtransfectionefficienciescanoftenyieldresults.Incontrast,forgeneknockdowntobemeasurablein

acellpopulationitisimportanttohavethehighesttransfectionefficiencypossible.Evensmallreductionsintransfectionefficiencycanlimityour

abilitytoidentifyfunctionaldifferencesinyourexperimentalsamplesorvalidateknockdownbyqRT-PCRorwesternblotanalysis.

Toachievethehighesttransfectionefficiencypossible,particularlyforgeneknockdownexperiments,firstoptimizetransfectionconditions

foryourcelllines,soyouknowyouhaveoptimaltransfectionconditionsforeffectiveRNAiexperimentation.Keepinmindthatitisalsoimportant

tomonitorexperiment-to-experimenttransfectionvariation.

We recommend three transfection reagents for delivering Silencer® Select siRNA or Stealth RNAi™ siRNA, Lipofectamine® RNAiMAX,

Lipofectamine®2000,andOligofectamine™reagents.Tomonitortransfectionefficiencyusingthesereagents,Invitrogenofferstwofluorescently

labeledRNAiduplexes: theBLOCK-iT™AlexaFluor®RedFluorescentControl forusewithLipofectamine®RNAiMAX,andthegreenBLOCK-iT™

FluorescentOligoforusewithLipofectamine®2000orOligofectamine™reagents(theBLOCK-iT™AlexaFluor®RedFluorescentControlcanalso

beusedwithLipofectamine®2000orOligofectamine™reagents).

Controls for RNAi experiments

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FormoreinformationoncontrolstouseforoptimizingtransfectionwithsyntheticRNAioratablelistingthecontentsforeachofthetrans-

fectioncontrolkits,visitwww.invitrogen.com/RNAicontrols.

InvitrogenalsorecommendstwotransfectionreagentsfordeliveringRNAivectorstocells:Lipofectamine®2000andLipofectamine®LTX

reagents.WealsorecommendtransfectingavectorthatexpressesafluorescentproteintomonitorcellularuptakeoftheRNAivector(BLOCK-iT™

PolIImiRRNAiExpressionVectorwithEmGFP).

FormoreinformationoncontrolstouseforoptimizingtransfectionwithRNAivectorsoratablelistingthecontentsforeachofthetransfec-

tioncontrolkits,visitwww.invitrogen.com/RNAivectorcontrols.

Negative controls

NegativecontrolsiRNAs—siRNAswithsequencesthatdonottargetanygeneproduct—areessentialfordeterminingtheeffectsofsiRNAdelivery

andforprovidingabaselinetocomparesiRNA-treatedsamples.TherearetwoSilencer®SelectNegativeControlsiRNAs.ThesesiRNAsincludethe

samemodificationsforreducingoff-targeteffectsasfoundinotherSilencer®SelectsiRNAsandhavenosignificantsequencesimilaritytomouse,

rat,orhumangenesequences.ThesenegativecontrolsiRNAshavebeentestedbymicroarrayanalysisandshowntohaveminimaleffectson

geneexpression. Inaddition, thesetwocontrolshavebeentested inmultiparametriccell-basedassaysandareproventohavenosignificant

effectoncellproliferation,viability,ormorphologyinthecelllinestested.

ByusingtheBLOCK-iT™RNAiDesigner,youcanchoosescrambledcontrolsforanyStealthRNAi™siRNAsequence.Wealsoprovideanega-

tive control in our RNAi vector cloning kits. The negative control should be the same chemical structure as the target RNAi molecules. For

example,ifyouareusingshRNAvectors,thenegativecontrolshouldhavethesamevectorbackbone,butadifferentRNAisequence.

ForexperimentsusingStealthRNAi™siRNA,wehavethreepredesignednegativecontrolswiththefollowingfeatures:

→ ThreelevelsofGCcontenttomatchthatoftheexperimentalStealthRNAi™siRNA

→ Nohomologytoanyknownvertebrategene

→ Testedsequences,whichdonotinduceastressresponse

WerecommendusingoneormorenegativecontrolineveryRNAiexperiment.

Positive controls

PositivecontrolsprovidemoreconfidenceinyourRNAiexperimentsbyensuringthattheexperimentalconditionsweremettoachieverobust

data.PositivecontrolsareRNAimolecules thatareknown toachievehigh levelsof knockdown (>70%).Apositivecontrol shouldbeused to

optimizeRNAideliveryconditionsandtoreconfirmhighlevelsofdeliveryineachRNAiexperiment.Whenapositivecontrolfailstoproducethe

anticipatedphenotype,carefullyevaluateyourexperimentalconditionsanddecideifsomefactorsneedtobeadjusted.Examplesofpositive

controlsaregenesexpressedateasilydetectablelevels,suchasp53,laminorGAPDH.However,ifyouarelookingataparticularphenotypesuch

asapoptosis,youwillmostlikelywanttochooseapositivecontrolknowntoelicitapoptosis,suchasEG5.

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Downstream controls

BeforetransfectingcellsandperformingqRT-PCRandwesternblotstomeasuremRNAandproteinlevels,werecommendvalidatingyourdown-

streamreagents.ValidatingqRT-PCRprimersorantibodiesforyourpositivecontrolandtargetgenesbeforeperformingknockdownexperiments

inyourcelllineensuresthatyourreagentsaresensitiveenoughtodetectchangesinexpressionofyourtargetgeneduetoknockdown.Without

sufficientsensitivity,itcanbedifficulttointerpretknockdownresultsfromgenesorproteinswithlowexpressionlevels.

Interferon controls

The introduction of RNAi reagents to cells can induce cellular stress response pathways, such as the interferon response. Activation of these

stressresponsepathwayscanleadtotranslationalarrest,growthinhibition,andcellulartoxicity.Theseeventsmakeitdifficulttoassesswhether

observedcellularphenotypesareduetononspecificstressresponsesorthelossoffunctionofthetargetedgene.ValidatedqRT-PCRprimersfor

PKR,IFIT-1and5’OASstressresponsegenesprovideaspecificandsensitivewaytomonitorwhethertoxiccellulareffectsarecomplicatingthe

interpretationofyourRNAiexperimentaldata.

Use multiple siRNA sequences per target to verify results

siRNA sequences with partial homology to other targets may contribute to off-target activity. Gene profiling experiments have shown that

duplexeswithpartialhomologytoothertranscriptscancleavethetargetoract likeamicroRNA(miRNA), inhibitingtranslationof thetarget

mRNA.SpecificitystudieshaverevealedthatsiRNAduplexescanhavevaryingactivitiesdependingonthenumber,position,andbasepaircom-

positionofmismatcheswithrespecttothetargetRNA.ToensurethatknockdownoftheintendedgenecausesaparticularsiRNAphenotype,

thephenotypicresultsshouldbeconfirmedbyatleasttwosiRNAmoleculesthattargetnon-overlappingregionsofthetargetmRNA.Thus,if

onesiRNAsequenceproducesaparticularphenotype,butthesecondsiRNAsequence(designedtotargetthesamegene)producesadifferent

phenotype,thenyoucannotconcludethatthegeneofinterestwassuccessfullyknockeddown.

Titrate siRNA

Silencer®SelectsiRNAandStealthRNAi™siRNAcanbeveryeffectiveevenatlowconcentrations.TitratingdownthedoseoftheSilencer®Select

siRNAandStealthRNAi™siRNAduplexenablesyoutoreduceanyoff-targetornonspecificeffectswhileachievingrobustknockdown.

Rescue experiments

RNAirescueexperimentsareperformedtoensurethattheobservedeffectisduetoknockingdownthetargetgeneofinterest.Ifyouareusing

aninducibleRNAivectorsystem,turnofftheRNAiexpressionbyremovingtetracyclinefromthemedium.IfyouareusingSilencer®SelectsiRNA

orStealthRNAi™siRNAtherearetwomainmethodsusedtorescuethephenotype.ThefirstmethodinvolvesdesigningRNAisequencestothe

3’UTRandthentransfectingthecellsafterknockdownwithavectorexpressingthetheopenreadingframe(ORF)ofthegeneofinterest.Ifthe

RNAisequencesweredesignedtotheORF,youcanuseamutagenesiskittocreateoneormoresilentthird-codonpointmutationswithinthe

regiontargetedbytheRNAisequence,preferablytheseedandcleavageregionsontheantisensestrand(bases2–12).


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Table 3.1. Recommended RNAi delivery methods.*

Cell type Transient expression (<7 days) Transient expression (>7 days) Stable expression

Fast-growingadherentcells(A549,HeLa)

LipidtransfectionofSilencer®SelectsiRNAorStealthRNAi™siRNA

LipidtransfectionofRNAivectorsoradenoviraldelivery

LipidtransfectionofRNAivectorsorlentiviraldelivery

Fast-growingsuspensioncells(THP-1)

LipidtransfectionorelectroporationofSilencer®SelectsiRNAorStealthRNAi™siRNA

LipidtransfectionofRNAivectorsoradenoviraldelivery

LipidtransfectionorelectroporationofRNAivectorsorlentiviraldelivery

Primarycells Lentiviraldelivery

Nondividingcells Lentiviraldelivery

*ForlipidtransfectionwerecommendLipofectamine®RNAiMAX(www.invitrogen.com/RNAiMAX),andforelectroporationwerecommendtheNeon™TransfectionSystem(www.invitrogen.com/neon).

CHAPTER 3

Delivering RNAi to cells—transfection and viral deliveryThetwocommonapproachesforRNAideliveryarelipid-mediatedtransfectionandviral-mediatedtransduction.Determiningwhichoneofthese

approachestousedependsonthecelltypebeingstudiedandwhethertransientorstableknockdownisdesired(Table3.1).Themostpopular

application,transienttransfectionofSilencer®SelectsiRNAsorStealthRNAi™siRNAduplexes,usescationiclipid–basedreagentsbecausetheyare

suitablefordeliveringmoleculesacrossadiverserangeofcommonlyusedcelllines.

Forcelltypesthatarenotamenabletolipid-mediatedtransfection,viralvectorsareoftenemployed.Adenoviralvectorsworkwellfortran-

sientdeliveryinmanycelltypes.However,whenstableRNAiexpressionisdesired,orfordifficultcelllines,suchasnondividingcells,lentiviralvec-

torsarethebestdeliverymethod.AnotherapproachfordeterminingthemostfavorableRNAideliveryconditionsistouseInvitrogen’sdelivery

optimizationservice—ascientificresourcewithextensiveknowledgeandexpertiseinviralvectorsandnonviraldeliveryreagentsfortestinga

matrixofdeliveryparameters.

Methods to achieve high transfection efficiency

TransfectionefficiencydescribesthepercentageofcellsthathavereceivedtheRNAiduplexorexpressionplasmid.Typically,researchersstriveto

achievethehighestlevelsoftransfectionefficiencypossible.ThisobjectiveisparticularlyimportantforRNAiapplicationsbecausenontransfected

cellswillcontinuetoexpressthegenetargetedforknockdown,thuscontributingtobackgroundexpressionlevels.

Formanydiseasemodels,themostdesirablecelltypesareprimarycultures. However,thesecannotbetransfectedadequatelywithcom-

merciallyavailablecationiclipid-mediatedtransfectionreagents.ApowerfulalternativeisviraldeliveryofvectorsexpressingRNAisequences.This

optionisrecommendedfordeliverytohard-to-transfect,primary,andnondividingcells.Viraldeliverycanalsobeusedtocreatestablecelllines

withinducibleRNAiexpressionortoexpressRNAisequenceswithtissue-specificpromoters.

Importance of minimizing transfection-mediated cytotoxicity

ThedeliveryofRNAireagents,orthedeliverymethoditself,cangiverisetocytotoxicityingenesilencingexperiments.Minimizingtransfection-

mediatedcytotoxicityisessentialforproperinterpretationoftheoutcomeofanyRNAiexperiment,ascytotoxiceffectscanbedifficulttodistin-

guishfromaphenotyperesultingfromtargetgeneknockdown.Cytotoxicityfromthedeliverymethoditselfislikelywhenapparentknockdown

ofthetargetgeneisseenwhencellsaretransfectedwithanegativecontrolwithsimilarGCcontenttothatofthetargetgene.Theeasiestway

tocombattheissueofdelivery-relatedcytotoxicityistochooseatransfectionreagentthathasbeendesignedforeitherdouble-strandedRNA

orplasmid-basedRNAitransfections.Mostoften,thesereagentshavebeenformulatedtomaximizeefficiency(toachievehighknockdownlev-

els)whileminimizingcytotoxicity.Optimizationexperimentsusingsupplierorpublishedprotocolsasguidelinescanhelptodeterminewhich

concentrationoftransfectionreagentworksbestforthecelllineofinterest.Werecommendusingthelowestamountoftransfectionreagent

necessarytoachievethehighestlevelofknockdown.

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Chapter 3Delivering RNAi to cells–transfection and viral delivery

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FEATURED PROTOCOL

Transfecting Stealth RNAi™ siRNA or Silencer® Select siRNA into A549 cells using Lipofectamine® RNAiMAX

IntroductionLipofectamine® RNAiMAX Reagent is a proprietary formulation specifically developed for highly efficient delivery of Stealth RNAi™ siRNA or

Silencer®SelectsiRNAtomammaliancellsforRNAianalysis.ThisreferenceprovidesarecommendedproceduretotransfectStealthRNAi™siRNAor

Silencer®SelectsiRNAintohumanA549lungcarcinomacells(ATCC,Cat.No.CCL-185)usingLipofectamine®RNAiMAX(Cat.Nos.13778-075,13778-

150).Lipofectamine®RNAiMAXhasabroadrangeofactivity,enablingmaximalknockdownlevelswithaminimumofoptimizationrequired.

Important guidelines for transfectionFollowtheseimportantguidelineswhentransfectingStealthRNAi™siRNAorSilencer®SelectsiRNAintoA549cellsusingLipofectamine®

RNAiMAX:

→ BothReverse transfectionandForward transfectionprotocols(page12)canbeusedfortransfectingA549cells.

→ To assess transfection efficiency, we recommend using a KIF11 Stealth Select RNAi™ siRNA, as described in Assessing transfection

efficiency(page12).

→ Werecommendusing10nMofthesiRNAduplexandindicatedprocedures.However,theefficacyoftheRNAisequencechosen,thetranscription

rateofthetargetgene,andthestabilityoftheresultingproteininfluencethedegreeoftargetgeneknockdownobserved.Youmayneedtoadjust

theRNAiconcentration(1–50nMcanbeused)andassaytime(upto72hours)toestablishoptimalknockdownofyourtargetgene.

→ WerecommendOpti-MEM®IReducedSerumMedium(Cat.No.31985-062)todiluteRNAiduplexesandLipofectamine®RNAiMAX

beforecomplexing.

→ Do notaddantibioticstomediaduringtransfectionasthiscausescelldeath.

→ Testserum-freemediaforcompatibilitywithLipofectamine®RNAiMAX.

→ Lipofectamine®RNAiMAXhasabroadpeakofactivity;forarangeofcelldensitiesandvolumesoftransfectionreagentsuitablefor

use,seeAcceptable range for maximal activity(page13).

Materials neededHavethefollowingreagentsonhandbeforebeginning:

→ A549cellsmaintainedinDMEM(Cat.No.11965-092)supplementedwith10%fetalbovineserum(Cat.No.26140-079),2mMglutamine

(Cat.No.25030-149),andpenicillin/streptomycin(Cat.No.15070-063)

Note:Uselow-passagecells(<50passages);makesurethatcellsarehealthyandgreaterthan90%viablebeforetransfection.

→ Silencer®SelectsiRNAorStealthRNAi™siRNAofinterest

→ Lipofectamine®RNAiMAXReagent(storeat+2–8°Cuntiluse)

→ Opti-MEM®IReducedSerumMedium

→ Appropriatetissuecultureplatesandsupplies

Significance of reducing off-target effects

Aswellasbeingasourceofcytotoxicity,asuboptimaldeliveryreagentorexcessreagentcanresultinapparentoff-targeteffects.Onecauseof

off-targeteffectsistheup-ordown-regulationofgenesduetothegenedeliveryprocedure.However,withappropriatecontrols,theseeffects

canbeidentifiedanddiminished.Again,usethelowestamountoftransfectionreagentthatprovidesthebestgenesilencingactivity.

Thepotentialalsoexistsforoff-targeteffectsduetoknockdownfromthesiRNAduplexitself.Todeterminethemostfavorableconditions,

varytheconcentrationofsiRNAwhileholdingtheconcentrationoftransfectionreagentconstantatthelowestconcentrationpreviouslyidenti-

fied.UtilizethelowestsiRNAconcentrationthatgivesthedesiredlevelofknockdowninRNAiexperiments.Keepinmindthatthespecificityof

thesiRNAwillhaveanimpactonpotentialoff-targeteffects.Theuseofexceptionallyspecificreagents,suchasSilencer®SelectsiRNAormodified

StealthRNAi™siRNAduplexes,canhelpalleviatetheseconcerns.

Delivering RNAi to cells–transfection and viral delivery3



FEATURED PROTOCOL, CONTINUED

Reverse transfectionUsethisproceduretoreverse-transfectStealthRNAi™siRNAorSilencer®SelectsiRNAintoA549cellsina24-wellformat(forotherformats,

seeScaling up or down transfections,page13).Inreversetransfections,thecomplexesarepreparedinsidethewells,afterwhichcellsand

mediumareadded.Reversetransfectionsarefastertoperformthanforwardtransfections,andarethemethodofchoiceforhigh-through-

puttransfection.Allamountsandvolumesaregivenonaperwellbasis.

1. Foreachwelltobetransfected,prepareRNAiduplex–Lipofectamine®RNAiMAXcomplexesasfollows:

a. Dilute6pmolRNAiduplexin100µLOpti-MEM®IMediumwithoutseruminthewellofthetissuecultureplate.Mixgently.

Note:IfthevolumeofyourRNAiduplexsolutionistoosmalltodispenseaccurately(lessthan1µL),andyoucannotpooldilutions,

prediluteyourRNAiduplex10-foldin1XRNAAnnealing/DilutionBuffer(orthedilutionbufferrecommendedbyyourRNAiduplex

manufacturer),anddispensea10-foldhigheramount(shouldbeatleast1μLperwell).Forexample,toget6pmolofRNAiduplex

froma20μMRNAiduplexstocksolution,diluteyourRNAiduplex10-foldtoaconcentrationof2μM,anddispense3µL.

b. MixLipofectamine®RNAiMAXgentlybeforeuse,thenadd1µLLipofectamine®RNAiMAXtoeachwellcontainingthediluted

RNAimolecules.Mixgentlyandincubatefor10–20minutesatroomtemperature.

2. DiluteA549cellsincompletegrowthmediumwithoutantibioticssothat500µLcontains30,000cells(celldensityshouldbe30–50%

confluent24hoursafterplating).

3. ToeachwellwithRNAiduplex–Lipofectamine®RNAiMAXcomplexes,add500µLofthedilutedcells.Thisgivesafinalvolumeof

600µLandafinalRNAconcentrationof10nM.Mixgentlybyrockingtheplatebackandforth.

4. Incubatethecells24–72hoursat37°CinaCO2incubatoruntilyouarereadytoassayforgeneknockdown.

Forward transfectionUsethisproceduretoforward-transfectStealthRNAi™siRNAorSilencer®SelectsiRNAintoA549cellsina24-wellformat(forotherformats,

seeScaling up or down transfections,page13).Inforwardtransfections,cellsareplatedinthewells,andthetransfectionmixisgenerally

preparedandaddedthenextday.Allamountsandvolumesaregivenonaperwellbasis.

1. Onedaybeforetransfection,plate30,000cellsin500µLofgrowthmediumwithoutantibiotics.Thecelldensityshouldbe30–50%

confluentatthetimeoftransfection.

2. Foreachwelltobetransfected,prepareRNAiduplex–Lipofectamine®RNAiMAXcomplexesasfollows:

a. Dilute6pmolRNAiduplexin50µLOpti-MEM®IReducedSerumMediumwithoutserum.Mixgently.

Note:IfthevolumeofyourRNAiduplexsolutionistoosmalltodispenseaccurately(lessthan1µL),andyoucannotpooldilutions,

prediluteyourRNAiduplex10-foldin1XRNAAnnealing/DilutionBuffer(ordilutionbufferrecommendedbyyourRNAiduplex

manufacturer),anddispensetheproperhigheramount(shouldbeatleast1µLperwell).Forexample,toget6pmolofRNAi

duplexfroma20μMRNAiduplexstocksolution,diluteyourRNAiduplex10-foldtoaconcentrationof2μM,anddispense3µL.

b. MixLipofectamine®RNAiMAXgentlybeforeuse,thendilute1µLin50µLOpti-MEM®IReducedSerumMedium.Mixgently.

c. CombinethedilutedRNAiduplexwiththedilutedLipofectamine®RNAiMAX.Mixgentlyandincubatefor10–20minutesatroom

temperature.

3. AddtheRNAiduplex–Lipofectamine®RNAiMAXcomplexestoeachwellcontainingcells.Thisgivesafinalvolumeof600µLanda

finalRNAconcentrationof10nM.Mixgentlybyrockingtheplatebackandforth.

4. Incubatethecells24–48hoursat37°CinaCO2incubatoruntilyouarereadytoassayforgeneknockdown.Mediummaybechanged

after4–6hours,butthisisnotrequired.

Assessing transfection efficiencyToqualitativelyassesstransfectionefficiency,werecommendusingaKIF11StealthSelectRNAi™siRNA(availablethroughwww.invitrogen

.com/rnaiexpress; for human cells, oligo HSS105842 is a good choice). Adherent cells in which KIF11/Eg5 is knocked down exhibit a

“rounded-up”phenotypeafter24hoursduetoamitoticarrest [1]; slowgrowingcellsmaytakeupto72hours todisplay therounded

phenotype.Alternatively,growthinhibitioncanbeassayedafter48–72hours.

Note:TheBLOCK-iT™FluorescentOligo(Cat.No.2013)isoptimizedforusewithLipofectamine®2000reagent,andisnotrecommended

forusewithLipofectamine®RNAiMAX.

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FEATURED PROTOCOL, CONTINUED

Acceptable range for maximal activityDuetothebroadrangeofmaximalactivityexhibitedbyLipofectamine®RNAiMAX,arangeofcelldensitiesandvolumesofLipofectamine®

RNAiMAXcanbeusedfortransfection.FortransfectingA549cellsin24-wellformat,0.5–1.25µLLipofectamine®RNAiMAXand20,000–

50,000cellsperwellissuitable.Forextendedtimecourseexperiments(72hours),considerusingthelowercellnumber;forshort-term

experiments(24hours),considerthehighercellnumber.ThefinalconcentrationofRNAiduplexcanbevariedbetween1and50nM.A

concentrationof10nMRNAiduplexissuitabletoknockdownmanytargetgenes.However,theoptimalconcentrationofRNAiduplexwill

varydependingontheefficacyoftheduplex,andshouldbedeterminedempirically.

Scaling up or down transfections TotransfectA549cellsindifferenttissuecultureformats,varytheamountsofStealthRNAi™siRNAorSilencer®SelectsiRNA,Lipofectamine®

RNAiMAX,cells,andmediumusedinproportiontotherelativesurfacearea,asshownbelow.

Note:20μMStealthRNAi™siRNAorsiRNA=20pmol/µL.

Recommended reagent amounts and volumes.

Cult

ure

vess

el

Rela

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sur

face

are

a*

Volu

me

of p

lati

ng m

ediu

m

Cells plated per well Dilution medium RNAi duplex amount

Final RNAi duplex concentration

Lipofectamine® RNAiMAX†

Star

t poi

nt

Acc

epta

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rang

e

Reve

rse

tran

sfec

tion

(µL)

Forw

ard

tran

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(µL)

Star

t poi

nt (p

mol

)

Acc

epta

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rang

e (p

mol

)

Star

t poi

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M)

Acc

epta

ble

rang

e (n

M)

Star

t poi

nt (µ

L)

Acc

epta

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rang

e (µ

L)

96-well 0.2 100µL 7,500 5,000–10,000 20 2x10 1.2 0.12–6 10 1–50 0.2 0.1–0.25

48-well 0.4 200µL 15,000 10,000–20,000 40 2x20 2.4 0.24–12 10 1–50 0.4 0.25–0.5

24-well 1 500µL 30,000 20,000–50,000 100 2x50 6 0.6–30 10 1–50 1 0.5–1.25

6-well 5 2.5mL 150,000 100,000–250,000 500 2x250 30 3–150 10 1–50 5 2.5–6.25

*Surfaceareasmayvarydependingonthemanufacturer.†IfthevolumeofLipofectamine®RNAiMAXistoosmalltodispenseaccurately,andyoucannotpooldilutions,prediluteLipofectamine®RNAiMAX10-foldinOpti-MEM®IReducedSerumMedium,anddispensea10-foldhigheramount(shouldbeatleast1.0µLperwell).DiscardanyunuseddilutedLipofectamine®RNAiMAX.

Want transfection protocols? Go to www.invitrogen.com/rnaitransfectionprotocol.




Cell health

In general, healthy cells take up nucleic acids better than poorly maintained cells. Many cells undergo expression profile changes that can

adverselyaffectyourexperimentswhentheyarestressedbycultureconditions.Routinelysubculturingcellsbeforetheybecomeovercrowded

orunhealthywillminimizeinstabilityincontinuouscelllines.InformationonbasiccellculturetechniquecanbefoundinCulture of Animal Cells,

a Manual of Basic Technique[2].

Culture conditions

Overlycrowdedorsparseculturesarenotconduciveforhealthycells.Asarule,cellsshouldbereplatedbeforethemediumbecomesdepleted.

Ascellculturesapproachconfluence,theytypicallycontainsomenumberofeitherunhealthyordeadcells,whichmakecellcountsinaccurate.

Inaddition, cells thathavegrown indepletedmediumbetweensubculturingeventshavebeendeprivedofnutrientsandmayhaveexperi-

encedpHshifts thataredetrimental tohealthandviability.Therefore,avoidovergrowingcellsandandsubjecting themto frequentpHand

temperatureshifts.

Someadherentcell linesaresensitivetotrypsinexposureandtoshearforcesfromvigorouspipettingorhigh-speedcentrifugation.(For

mostcelllines,trypsinizationshouldbekeptshorterthan10minutes.)Inadditiontotreatingcellsgently,maintainingstrictprotocols,including

harvestingcells forexperimentsat similarconfluenciesandmaintainingconsistent time intervalsbetweenplatingand transfectingcells,will

improveexperimentalreproducibility.

Mycoplasmacontaminationisanothercommonstresstocellsinculturethatcandeleteriouslyeffectexperimentalresults.Mycloplasmaare

small,free-livingprokaryotesthatarenotobservablebylightmicroscopy.Becausetheygrowasfilamentousorcoccalfromswithoutcellwalls,

theyarenotsensitivetoantibioticsthatinterferewithcellwallproduction.Mycoplasmascanaltercellgrowthcharacteristics,inhibitcellmetabo-

lism,anddisruptnucleicacidsynthesis,causingchromosomalabnormalities,andalteringtransfectionorinfectionrates.Inmostsituations,myco-

plasmasfromaninfectedcelllinespreadtootherculturesinalaboratoryviaaerosolizationduringroutinepipettingandhandlingorfromshared

reagents(e.g.,medium,serum)thatbecomecontaminated.Thebestpreventionandcontrolrequiresgoodaseptictechnique(includingworking

withculturesinorderofcleantountestedtoinfectedduringtheworkdayorweek)androutinetesting.Manycommercialkits(PCR-,ELISA-,fluo-

rescence-,luminescence-,andculture-basedassays)areavailabletotestculturesformycoplasmacontamination.Culturesinfectedwithmyco-

plasmasareusuallydiscardedandreplaced,butforirreplaceablecultures,treatmentoptionsareavailabletoinhibitoreliminatemycoplasmas.

TIPS→ Let freshly thawed cells recover for at least 48 hours.Donotperformanalysesonfreshlythawedcellswithin48hoursofplating.

→ Optimization of siRNA delivery for different phenotypic assays. SimilartobalancingsiRNA-inducedknockdownandcellviability,

theremayalsobeabalancebetweensiRNAdeliveryanddownstreamassayconditions. ItmaybenecessarytoreoptimizesiRNA

deliveryconditionsfordifferentdownstreamassaysthatareusedinsiRNAscreeningpasses.

Passage number

Becausesomecelllinesmaygraduallychangeinculture,werecommendusingnormalorprimarycelltypeswithin10passagesofdetermining

optimalsiRNAdeliveryconditions.Iftransfectionorelectroporationefficiencybeginstodrop,thawfreshcellsforsubsequentexperiments.Iffro-

zenstocksarenotavailableforreculturing,itmaybenecessarytoreoptimizethetransfectionconditionsusingexistingstocks.Passagenumber

isusuallynotascriticalforimmortalizedortransformedcelllines.

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siRNA quality

ThequalityofsiRNAcansignificantlyinfluenceRNAiexperiments.siRNAsmustbefreeofreagentscarriedoverfromsynthesis,suchasethanol,salts,and

proteins.Also,dsRNAcontaminantslongerthan30bpareknowntoaltergeneexpressionbyactivatingthenonspecificinterferonresponseandcausing

cytotoxicity[3].Therefore,werecommendusingsiRNAsthataregreaterthan80%fulllength(standardpuritysiRNAs).

siRNA quantity

TheoptimalamountofsiRNAanditscapacityforgenesilencingareinfluencedinpartbypropertiesofthetargetgeneproducts,includingthe

following:mRNAlocalization,stability,abundance,aswellastargetproteinstabilityandabundance.AlthoughmanysiRNAexperimentsarestill

performedbytransfectingcellswith100nMsiRNA,publishedresultsindicatethattransfectinglowersiRNAconcentrationscanreduceoff-target

effectsexhibitedbysiRNAs[4,5].Forlipid-mediatedreversetransfections,10nMofsiRNA(range1–30nM)isusuallysufficient.ForsiRNAdelivery

usingelectroporation,siRNAquantityhasalesspronouncedeffect,buttypically1μg/50µLcells(1.5μM)ofsiRNA(range,0.5–2.5μg/50µLcells

or0.75–3.75μM)issufficient.

KeepinmindthatwhiletoomuchsiRNAmayleadtooff-targetorcytotoxiceffects,toolittlesiRNAmaynotreducetargetgeneexpression

effectively.Becausetherearesomanyvariablesinvolved,itisimportanttooptimizethesiRNAamountforeverycelllineused.Inaddition,the

amountofnontargetingnegativecontrolsiRNAshouldbethesameastheexperimentalsiRNAs.

Choice of transfection agent

Itisimportanttoselecttheappropriatetransfectionagentforthecelllinebeingused.Differentcelltypesvaryintheirresponsetodifferenttrans-

fectionagents;thus,thebesttransfectionagentforaparticularcelltypemustbedeterminedexperimentally.Lipofectamine®RNAiMAXisknown

tobebestperformingsiRNAtransfectionreagentonthemarket.Formoreinformation,seeChapter9.

Volume of transfection agent

Thevolumeoftransfectionagent isacriticalparametertooptimizebecausetoo littlecan limittransfection,andtoomuchcanbetoxic.The

overall transfectionefficiency is influencedbytheamountoftransfectionagentcomplexedtothesiRNA.Tooptimize,titratethetransfection

agentoverabroaddilutionrange,andchoosethemostdiluteconcentrationthatstillgivesgoodgeneknockdown.Thiscriticalvolumeshould

bedeterminedempiricallyforeachcellline.

Whilecelldensityisimportantfortraditional,pre-platedtransfectionexperiments,celldensityislesscriticalandrequireslittletonooptimization,

whensiRNAsaredeliveredbyreversetransfection.However,iftoomanycellsareused,andtheamountofsiRNAisnotincreasedproportionally,

theconcentrationofsiRNAinthesamplemaybetoolowtoeffectivelyelicitgenesilencing.Whencelldensityistoolow,culturescanbecome

unstable.Instabilitycanvaryfromwelltowellbecausecultureconditions(e.g.,pH,temperature)maynotbeuniformacrossamultiwellplateand

candifferentiallyinfluenceunstablecultures.

Exposure to transfection agent/siRNA complexes

Althoughmosttransfectionagentsaredesignedtoinduceminimalcytotoxicity,exposingcellstoexcessiveamountsoftransfectionagentor

foranextendedtimecanbedetrimentaltotheoverallhealthofthecellculture.Sensitivecellsmaybegintodiefromexposuretothetransfec-

tionagentafterafewhours.Iftransfectioncausesexcessivecelldeathwithyourcells,removethetransfectionmixtureandreplenishwithfresh

growthmediumafter8–24hours.




Presence of serum in the medium during transfection

ComplexformationbetweentransfectionagentsandsiRNAshouldbeperformedinreduced-serumorserum-freemedium,sothatserumcom-

ponentswillnotinterferewiththereaction.However,oncecomplexformationhasoccurred,sometransfectionagentswillpermittransfectionin

serum-containing,normalgrowthmedium(followmanufacturer’sinstructions).Noculturemediumadditionorreplacementisusuallyrequired

followingtransfection,butchangingthemediacanbebeneficial insomecases,evenwhenserumcompatible reagentsareused.Besure to

checkforserumcompatibilitybeforeusingaparticularagent.Sometransfectionagentsrequireserum-freemediumduringthetransfectionand

achangetocompletegrowthmediaafteraninitialincubationwithtransfectioncomplexes.

Optimizing transfection experiments

Maximizing transfection efficiency while minimizing cytotoxicity are crucial for optimal gene silencing. The best transfection efficiencies are

achievedforeachcelltypebyidentifying(inorderofimportance):

1. Choiceoftransfectionreagent

2. Volumeoftransfectionagent

3. AmountofsiRNA

4. Celldensityatthetimeoftransfection

5. Lengthofexposureofcellstotransfectionagent/siRNAcomplexes

6. Transfectionmethod:traditionaltransfectionwherecellsarepre-platedorreversetransfectionwherecellsaretransfectedastheyadhere

totheplate

7. Presenceorabsenceofserum

Oncetheconditionsformaximalgenesilencingaredetermined,keepthemconstantamongexperimentswithagivencelltype.

TIPS→ siRNA storage:StoresiRNAsat–20°Cor–80°C,butdonotuseafrost-freefreezer.Ourdataindicatethatupto50freeze/thawcycles

arenotdetrimentaltosiRNAsinsolutionat100μM(asassessedbymassspectrometryandanalyticalHPLC).However,werecommend

thatsiRNAsthathavebeenresuspendedinRNase-freewaterorbufferbestoredinsmallaliquotstoavoidpotentialcontamination.

→ Nuclease resistance of siRNAs:Annealed,double-strandedsiRNAsaremuchmorenuclease resistant than single-strandedRNA.

However,stringentRNase-freetechniquesshouldbeusedduringallRNAiexperiments.

→ Checking siRNA for degradation:IfyoususpectthatapreparationofsiRNAmaybedegraded,checktheintegrityofthesiRNAby

running~2.5μgonanondenaturing15–20%acrylamidegel.VisualizetheRNAbystainingwithethidiumbromide,andverifythatit

istheexpectedsizeandintensity.ThesiRNAshouldmigrateasatightband;smearingindicatesdegradation.

Chapter references1. WeilDetal.(2002)Biotechniques33:1244–1248.

2. FreshneyRI(2000)4thed.NewYork(NY):Wiley-Liss.

3. StarkGRetal.(1998)Ann Rev Biochem67:227–264.

4. JacksonALetal.(2003)Nat Biotechnol21(6):635–637.

5. SemizarovDetal.(2003)Proc Natl Acad Sci U S A100(11):6347–6352.

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Chapter 4Vector-based RNAi technologies

www.invitrogen.com

No treatment Lenti-miR-negative

Lenti-miR-MAP2A

Figure 4.1. Lentiviral transduction of miR RNAi. Untreated(A, B)andtransduced(C–F)sampleswerestainedwithMAP2antibodies(orange)andDAPI(blue).EandFclearlyshowlesstargetproteinexpressioncomparedtotheuntreatedneuronsandthosetransducedwiththeLenti-miR-negativecontrol.

CHAPTER 4

Vector-based RNAi technologies

Introduction to adenoviral and lentiviral RNAi vectors for delivery

Havingevolvedtoproficientlydelivernucleicacidstocells,virusesofferameanstoreachhard-to-transfectcelltypesforproteinoverexpression

or knockdown. Adenoviral, oncoretroviral, and lentiviral vectors have been used extensively for delivery in cell culture and in vivo (Table 4.1).

AdenovirusesareDNAvirusesthatcantransientlytransducenearlyanymammaliancelltype,includingnondividingprimaryandgrowtharrested

cells.AdenovirusesentertargetcellsbybindingtotheCoxsackieadenovirusreceptor(CAR).AfterbindingtotheCAR,theadenovirusisinternal-

izedvia integrin-mediatedendocytosis, followedbyactivetransport tothenucleus,where itsDNA isexpressedepisomally.Oncoretroviruses

and lentivirusesarepositive-strandRNAvirusesthatstably integratetheirgenomes intohostcellchromosomes.Whenpseudotypedwithan

envelopethathasabroadtropism,suchasvesicularstomatitisvirusglycoprotein(VSV-G),thesevirusescanentervirtuallyanymammaliancell

type.However,theoncoretrovirusesdependuponnuclearmembranebreakdownduringcelldivisiontotransducecells.Incontrast,lentiviruses

aremoreversatiletools,astheyuseanactivenuclearimportpathwaytotransducenondividingcells(Figure4.1).

Table 4.1. Viral delivery strategies for RNAi.

Transient expression Stable expression

Dividingcells

Nondividingcells

Dividingcells

Neuronalcells

Drug-orgrowth-arrestedcells

Contact-inhibitedcells

Adenovirus • •

Lentivirus • • • • • •

Oncoretrovirus • •

Vector-based RNAi technologies4



Store up to 1 year

Transduce mammaliancells, select for stableexpression, and assay

ViraPower™ 293FT producer cell line

Generate the pLenti expression construct containing the miR RNAi

or shRNA of interest

Combine with optimizedViraPower™

Packaging Mix

Cotransfect the ViraPower™ 293FT producer cellline with the pLenti expression construct and

the optimized ViraPower™ Packaging Mix

Harvest viral supernatantand determine the titer

pLenti/BLOCK-iT™

RNAiconstruct

Ampicillin SV40 pA

RRE

pUC ori

PSV40

PRSV

/5´ L

TR

∆U3/

3´ L

TR

ψ

EM7

Selection

marker

Table 4.2. Choose a lentiviral or adenoviral RNAi system.

Viral system When to use Products

LentiviralRNAideliverysystems

• StableRNAiinanycellline,evennon-dividingcells

• InducibleorconstitutiveshRNAormiRRNAiexpression

• Studiesinanimalmodels

• BLOCK-iT™LentiviralPolIImiRRNAiExpressionSystem—acompletelentiviralsystemwithalloftheadvantagesofmiRRNAi:multiple-targetknockdownandahigherdesignsuccessratethanconventionalshRNA(containspLenti6/V5-DEST™vector)

• BLOCK-iT™LentiviralPolIImiRRNAiExpressionSystemwithEmGFP—asystemwithallofthebenefitslistedabove,pluseasyexpressiontrackingwithcocistronicEmGFP(containspLenti6/V5-DEST™vector)

• BLOCK-iT™InducibleH1LentiviralRNAiSystem—completelentiviralsystemforinducibleorconstitutiveshRNAexpressioninanycelltype(containspLenti4/BLOCK-iT™-DESTvector)

• BLOCK-iT™LentiviralRNAiExpressionSystem—completelentiviralsystemforconstitutiveshRNAexpressioninanycelltype(containspLenti6/BLOCK-iT™-DESTvector)

BLOCK-iT™RNAiAdenoviralSystem

• High-leveltransientshRNAexpression• Effectivedeliverytoawiderangeof

humancelltypes• Studiesinanimalmodels

• BLOCK-iT™AdenoviralRNAiExpressionSystem—completesystemforhigh-leveltransientexpressionofshRNA

Lentiviral and adenoviral RNAi vectors—choose any cell type for RNAi

Formanydiseasemodels,themostdesirablecelltypes,suchasimmunesystemorprimarycells,arenotamenabletotransfection.Viraldelivery

ofRNAivectorsisapowerfulalternativetotransfectionforthesecelltypesaswellasforin vivo applications.Toaccuratelydeterminetheefficacy

ofknockdownfromanshRNA/miRRNAimoleculeinapopulationofcells,itiscriticaltodelivertheshRNA/miRRNAimoleculetoasmanycellsas

possible.Otherwise,whenknockdownismeasuredbyquantitativereal-timePCR(qRT-PCR)orwesternblotanalysis,thebackgroundofmRNAor

proteininnontransfectedcellswillmaketheknockdownappearlesseffectivethanitactuallyis.Viraldeliverycanbethebestoptioninvirtually

anymammaliancelltype,includinghard-to-transfect,primary,andevennondividingcells.Conveniently,lentiviraldeliverysystemsareavailable

forbothshRNAandmiRRNAivectors,andanadenoviraldeliverysystemisavailableforshRNAvectors(Table4.2).

TheprocedureforusingbothRNAiviralsystems(Figure4.2):

1. Clonethedouble-strandedDNAoligoencodinganshRNAormiRRNAiintooneoftheBLOCK-iT™entry(shRNA)orexpression(miRRNAi)

vectors.

2. TransfertheRNAicassetteintotheadenoviral(shRNAonly)orlentiviraldestinationvectorbyGateway®recombination.

3. Transfectvectors intotheappropriatepackagingcells (useViraPower™PackagingMixfor lentiviralsystemsonly)toproduceviralstocks,

whichcanbeusedimmediatelyorstoredat–80°C.

4. Harvestand(foradenovirusonly)amplifytheviralsupernatantanduseitforshRNA/miRRNAideliverytoanycelltype.

Figure 4.2. How the BLOCK-iT™ lentiviral RNAi systems work.

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Chapter 5In vivo RNAi

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21-mer siRNA Stealth RNAi™ siRNA 21-mer siRNA Stealth RNAi™ siRNA

Mouse serum Human serum

0 4 8 24 48 720 4 8 24 48 720 4 8 24 48 72 0 4 8 24 48 72

Figure 5.1. Chemical modification makes Stealth RNAi™ siRNA ideal for in vivo applications. StealthRNAi™siRNAduplexesarechemicallymodifiedtoenhancestabilityagainstnucleasesinserum.Unmodified21-merdsRNAsequencesandcorrespondingStealthRNAi™siRNAsequenceswereanalyzedat0,4,8,24,48,and72hrfollowingincubationineithermouseorhuman10%serum.SampleswereseparatedonaNovex®15%TBE-ureapolyacrylamideprecastgelandstainedwithmethyleneblue.

CHAPTER 5

In vivo RNAi

RNAi molecules for in vivo applications

Theobstaclesandchallengesforin vivo RNAideliveryareverydifferentfromthoseinin vitrosettings.Toachievesuccessfulknockdown, in vivo

siRNAhastosurviveopsonizationanddegradationbynucleases,targetparticularcells,andtrafficintotheappropriatecellcompartment.This

chapterissetuptoprovideguidelinesandprotocolsenablingsuccessfulin vivo RNAiexperiments.

Choosing RNAi effector molecules

siRNA vs. RNAi vectorsRNAicanbedeliveredusingtwodifferentapproaches:siRNAsyntheticduplexes,orsiRNAexpressedfromplasmidsorviralvectors(shRNA,miR

RNAi).siRNAisbecomingthemethodofchoiceforthefastdevelopmentoftherapeutics.Theyareeasytodesign,synthesize,anduse.siRNAcan

berapidlyidentifiedandmultiplegenescanbetargetedatthesametime.RNAivectorsoffersteadierexpressionbecausethevectorscantarget

nondividingstemcells,lymphocytes,andneurons.Additionally,thereisthepossiblityofstableintegrationofthesiRNAplasmidintothegenome

ofthetargetedcell.ThedrawbacksofRNAivectorsincludedangerofoncogenictransformationfrominsertionalmutagenesis,andunanticipated

toxicityfromlong-termsilencingofhumangenesand/orhighamountsofsiRNAinsidethecell[1].

Chemically modified vs. unmodified siRNAsDeliveredin vivo,standardsiRNAisrapidlydegradedandclearedfromplasmawithahalf-lifeofminutes[2].Chemicalmodificationsthatprolong

siRNAhalf-life,withoutjeopardizingbiologicalactivity,arehighlydesirableforsuccess in vivo.StealthRNAi™siRNAsarechemicallymodifiedRNA

duplexesare25basepairsinlengthwithbluntends.Theyofferhigherstabilityinserumforlonger-lastingknockdowneffectsincells(Figure5.1).

ModificationsonthesensestrandensurethatonlytheantisensestrandisutilizedbytheRNA-inducedsilencingcomplex(RISC)toinhibitatarget

In vivo RNAi5



Figure 5.2. Stealth RNAi™ siRNA avoids stress response. (A)TailveinsofBalb/cmicewere injectedwith50μgofeitherssDNAwithaCpGmotif,a37bpstandardsiRNA,orStealthRNAi™siRNAduplex. (B)After6hr, serumwascollectedandprocessedtomeasureIFNmarkersusingLuminex®multiplexassays.

→ No treatment

3 mice per treatment:

6 hrSacri�ce andcollect serum

Luminex®

assays

→ CpG in PBS (positive control)

→ 37 bp dsRNA

→ Stealth RNAi™ siRNA in PBS

IL-1αIL-1βIL-2IL-4IL-5IL-6IL-10IL-12p40IL-12p70IL-13IL-17TNF-αIFN-γ

G-CSFGM-CSFFGF-basicVEGFPDGF-BBMIP-1αMIP-1βMIP-3βKCIP-10MCP-1MIGRANTES

Cytokine 10-plexTh1/Th2 6-plexIn�ammatory 4-plexChemokine 5-plexGrowth factor 4-plexCytokine 20-plex

19 markers Multiplex panelsA. Protocol

Rel

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(log

sca

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0.1

1

10

100

37 bpdsRNA

2.5 mg/kg

Stealth RNAi™

siRNA2.5 mg/kg

CpGNotreatment

IL-6

IP10

MIP-1α

IL-12

MCO-1

TNF-α

B. Results

Choosing RNAi purity

Invitrogen’sproductionofin vivoRNAiduplexesbeginswithstandardsynthesisofRNAioligosusinghigh-qualitystartingmaterials(Figure5.3).All

RNAioligosarethenduplexedanddesalted.AtthispointtheresearchercanalsorequestHPLCpurification.However,thisstepaddscosttothe

processandreducesyield.Subsequentin vivopurityprocessingofRNAiduplexesincludesaseriesofdialysisandcounterionexchangestepsto

removetoxicsaltsandsolventsandlowertheconductivitytomatchphysiologicalconditions.Theresultinghigh-qualityduplexesarereadyfor

in vivouseregardlessofwhetherHPLCpurificationisrequestedupstreamofthisprocess:

1. HPLC—standardRNAioligosynthesisfollowedbyHPLCpurification

Advantage:Canbeconjugatedwithdyes

Disadvantage:Doesnotincludeextrasaltandsolventremoval

2. Desalted, in vivo purity—standard RNAi oligo synthesis followed by diafiltration to remove salts and solvents to a level of <200 µS and

sterilefiltration

Advantages:Availableathighscale(withoutcustomorder),offersimportantsaltandsolventremoval,mostcost-effective

Disadvantage:Cannotbemodifiedwithdyes

3. HPLC, in vivopurity—standardRNAioligosynthesis followedbyHPLCpurification,diafiltrationtoremovesaltsandsolventstoa levelof

<200µS,andsterilefiltration

Advantage:Highestpurityavailable

Disadvantage:Leastcost-effectiveduetoloweryields

RNA.Thisdecreasesthepotentialforoff-targeteffectssincethesensestrandcannotcleaveunintendedtargets.Additonally,siRNAhasthe

potentialtoactivatetheinnateimmuneresponse,settingoffdefensesystemsusuallyusedtocombatviruses.Thepresenceofthechemical

modificationsinStealthRNAi™siRNAsabolishtheimmunostimulatoryresponse(Figure5.2)observedwithsomesequences.

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Figure 5.3. Invitrogen produces high-quality in vivo RNAi duplexes in three purity selections supporting Alexa Fluor® dye and biotin modifications.

Standard synthesis

HPLCpuri cation

in vivoIn vivo purity processes• Diafiltration (<200 µS/cm)

• Sterile filtration

• Endotoxin testing (<2 EU/mg)Desaltedin vivo

HPLCpuri cation

Purity Modi cations

• HPLC • Alexa Fluor® 488

• Desalted in vivo • Alexa Fluor® 546

• HPLC in vivo • Alexa Fluor® 555

• Alexa Fluor® 647



• Biotin

Raf

-1/G

AP

DH

rat

io

Raf-1 +BLOCK-iT™

Raf-1 biotinRaf-1 Alexa Fluor® 647

Raf-1Control

0.2

0

0.4

0.6

0.8

1.0

1.2

1.4

Figure 5.4. Labeled Stealth RNAi™ siRNA exhibits potent knockdown. MDA-MB-435cellsweretransfectedwith25nmolunlabeledStealthRNAi™siRNAduplexes,orStealthRNAi™siRNAduplexeslabeledwithanAlexaFluor®dyeorbiotin,ineachcasetheduplexestargetingRaf-1.Knockdownwasassessedat>90%byqPCR;deliveryisdemonstratedthroughfluorescenceimaging.

Figure 5.5. An alternate method to show knockdown. Mixing unlabeled duplexwithlabeledcontrolduplexes.

Raf-1 RNAi–Alexa Fluor® 647Raf-1 RNAi, biotinylatedAlexa Fluor® 488–streptavidin

Raf-1 RNAi + BLOCK-iT™ Alexa Fluor® 555

Tracking delivered duplexes

We have demonstrated that labeling Stealth RNAi™ siRNA duplexes does not hamper their knockdown potency (Figure 5.4). An alternative

approach is tomixunlabeledduplexeswith labeledcontrolduplexes (Figure5.5); thismethod ismorecommonlyusedwhenperforming in

vivoRNAiandallowstheprogressiontoclinicalresearchtoproceedunhinderedbyquestionsaboutthepossibleeffectsofadyelabel.Tracking

optionsare:

→ Stealth RNAi™ siRNA can be combined with bright BLOCK-iT™ fluorescent controls (available in different scales, and colors shown in

Figure5.3)withoutlossofactivity

→ StealthRNAi™siRNAcanbedirectlylabeledwithAlexaFluor®dyes,withoutlossofactivity

→ StealthRNAi™siRNAcanbebiotinylatedandcombinedwitheitherstreptavidin-AlexaFluor®orstreptavidin-Qdot®labels

In vivo RNAi5



IN VIVO RNAi WITH VECTORSAlthoughemployingRNAivectorsystemscanbeslightlymoreinvolvedthanusingsyntheticRNAireagents,theflexibilityofthevector-

basedsystemsiscompellingformanyRNAiresearchersconductingboth in vitroand in vivoexperiments.Therearetwomaintypesof

RNAiexpressionvectortechnologiesonthemarket:shorthairpin(shRNA)expressionvectorsandartificialmicroRNA(miRNA)expression

vectors.MostRNAivectorsavailable todayemploy shRNAvector technology,which typically involves shRNAexpression fromaPol III

promoterandmayormaynotemployviraldelivery.ThesevectorsexpressshRNAsequences,typicallyfromaU6orH1promoter,and

somehaveinduciblepromoters(typicallyH1/TO—atetracycline-induciblepromoter).Whilethesevectorscanbeusedfor in vivoRNAi

experiments,therearesomedrawbacks,includinglowdesignsuccessrateandinabilitytotrackshRNAexpressionorexpresstheshRNA

inaspecifictargettissue.

RNAi vector delivery methodsSimilartoRNAivectorsforin vitroapplications,youcanuseeitherstandardtransfectiontechniquesoraviraldeliverymethodtodeliver

RNAivectorsin vivo.Currently,thedeliveryofanRNAiexpressionvector in vivowithoutusingaviraldeliverysystemissimilartodeliver-

ingsyntheticdsRNA in vivo.Typically, thiswould involvecomplexingtheRNAiexpressionvectorwithacommonlyused lipid-based in

vitro transfectionreagentandinjectingdirectlyintotheanimal.WhilethismaybetheeasiestapproachfordeliveringRNAivectorsinto

animals,ithasquiteafewlimitations,includingtheinabilitytodeliversystemically,andlowtransfectionefficiencies.Forthesereasons,

most researcherschoose touseaviraldeliverymethodwhenemployingRNAivectors for in vivoexperiments.Regardlessofwhether

onechoosesanshRNAormiRRNAivector system,viraldelivery isahugeadvantage formany in vivo approaches.Mostviraldelivery

approachesinvolveeitheradenoviral,retroviral(nonlentiviral),orlentiviraltechnology:

→ AdenoviruscanbeusedfortransientRNAiexpressionineitherdividingornondividingcells

→ Retroviruscanbeemployedfortransientorstableexpression,butcanonlybeusedtotransducedividingcells

→ Lentiviraldeliveryaffordsthemostoptions,asitcanbeusedfortransientorstableexpressionindividingornondividingcells,aswell

asneuronalcells,drug-orgrowth-arrestedcells,orevenprimarycells

Forprotocolsandfurtherrecommendations,seewww.invitrogen.com/invivornai.

In vivo RNAi protocols

Refertothefollowingsupplementalprotocolsforspecialconsiderationforin vivoexperiments.Forquestionsaboutaprotocol,pleasecontact

InvitrogenTechnicalSupporttohavearepresentativeguideyouthroughtheprocedure.

Protocolsforsuccessfulin vivoRNAiexperiments:

→ ResuspensionofStealthRNAi™siRNAfor in vivoapplications

→ MeasuringRNAconcentration

→ Tissueharvest/RNAextraction

→ Tissuesectioning

→ Proteinextractionfromtissues

→ Tissuepreparationforflowcytometryanalysis

Table 5.1. Overview of products for in vivo applications. Exploreourfulllineofin vivoRNAiresourcesatwww.invitrogen.com/invivornai.

RNA Purification Scale Alexa Fluor® dye Biotin

BLOCK-iT™siRNAandStealthRNAi™siRNA

Desalted+in vivopurity 25nmol,100nmol,2μmol NA NA

HPLC 5,20,or500nmol 488,546,555,647,680,750 Yes

HPLC+in vivopurity 5,20,or500nmol 488,546,555,647,680,750 Yes

BLOCK-iT™FluorescentOligoControlHPLC 5,20,or500nmol 488,546,555,647,680,750 Yes

HPLC+in vivopurity 5,20,or500nmol 488,546,555,647,680,750 Yes

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In vivo–purityStealthRNAi™siRNAduplexesarespecificallyformulatedforuseinanimals.ResuspendtheRNAduplexinUltraPure™DNase/

RNase-freedistilledwaterorappropriateDNase/RNase-freebuffer(e.g.,PBS,Ringer’ssolution,0.9%NaCl).A5mg/mLstocksolutionisrecom-

mendedfor in vivoRNAiexperiments.Table5.2andTable5.3specifytherecommendedresuspensionvolumefor in vivo–purityStealthRNAi™

siRNAandBLOCK-iT™siRNA.

NOTE: The recommended resuspension volumes are for an RNAi molecule with 50% GC content. The molecular weight of RNAi molecules varies slightly depending on the

GC content, but these differences are negligible for in vivo RNAi experiments.

Table 5.2. Desalted in vivo purity: recommended resuspension volume for 5 mg/mL final concentration.

Desalted in vivo quantity Stealth RNAi™ siRNA resuspension volume—in vivo purity siRNA resuspension volume—in vivo purity

25nmol 80 µL 67µL

100nmol 320 µL 260 µL

2μmol 6.4mL 5.4mL

Table 5.3. HPLC purity: recommended resuspension volume for 5 mg/mL final concentration.

HPLC-purified delivered quantity Stealth RNAi™ siRNA resuspension volume— in vivo purity siRNA resuspension volume—in vivo purity

5nmol 16 µL 13 µL

20 nmol 64 µL 53 µL

500 μmol 1.6 mL 1.3 mL

Measuring RNA concentration

Ifdesired,measureRNAconcentrationusingUVabsorbanceat260nm(A260).DilutetheRNAsolutioninresuspensionbufferorwater,mixwell.

MeasuretheA260ofthedilutioninaspectrophotometerblankedagainstdilutionbuffer(usingacuvettewitha1cmopticalpathlength).Calculate

theRNAconcentrationusingtheappropriateformula:

Stealth RNAi™ siRNA formulaRNAconcentration(μg/mL)=A260(OD260units)x44((μg/mL)/ODunit)xdilutionfactor

siRNA formulaRNAconcentration(μg/mL)=A260(OD260units)x41((μg/mL)/ODunit)xdilutionfactor

NOTE: The formulas for Stealth RNAi™ siRNA and siRNA are slightly different due to chemical and size differences.

In vivo RNAi5



Harvesting tissue—RNA extraction from tissue

1. Homogenize50–100mgoftissuein1mLTRIzol®PlusReagentusinglysingmatrixDontheFastPrep®-24Instrument(MPBiomedical)at4°C.

Atissuehomogenizerorrotor-statorcanalsobeused.

2. Forhardertissues(tumors,lungs),perform3cyclesof60secondseachat6m/susingatissuehomogenizerorrotorstator.Forsoftertissues

(brain,liver),1cycleof60secat4.5m/sissufficienttocompletelydissociatethetissue.Add0.2mLofchloroformdirectlyintothetubeand

processfollowingtheprotocoldescribedinthePureLink™RNAMiniKitPurificationSystemmanual.

3. DeterminethequantityandpurityofthepurifiedRNAusingUVabsorbanceat260nmorQuant-iT™RNAAssayKit(Cat.No.Q33140).To

determineRNAquality,electrophoreseonanE-Gel®agarosegeloranalyzeontheAgilent2100Bioanalyzer.

4. Afterquantification,use750ngoftotalRNAforfirst-strandsynthesisusingtheSuperScript®VILO™KitandqPCRanalysisperformedusing

TaqMan®GeneExpressionAssaystomeasureknockdown.

Sectioning tissue

Slide preparationIf you are preparing your own slides, precoat slides with HistoGrip™ Concentrate (Invitrogen Cat. No. 008050) or 0.1% poly-L-lysine in water,

thenair-dry.Commerciallyavailableprecoatedglassslidesareavailableandcanbeusedtomountfrozenorformalin-fixed,paraffin-embedded

tissuesections.

Frozen tissueAprotocolforpreparingfrozentissuesamplesisdescribedbelow,butthisisonlyaexample.Ifyourlaboratoryalreadyhasanoptimizedprotocol

foryoursampletype,werecommendthatyouusethatprotocol.

1. SnapfreezefreshtissuesincryomoldscontainingOCT®(OptimalCuttingTemperature)compound(asolutionofglycolsandresinswhich

providesaninertmatrixforsectioning).Storefrozentissueblocksat–70°Cuntilyouarereadyfortissuesectioning.

2. Forsectioning,allowthefrozentissueblocktoequilibratetothecryostattemperature,cut4–20µmcryostatsections,andmountoncoated

glassslides.

3. Drytissuesectionsatroomtemperaturefor30minutes.

4. Ifdesired,storeslidesat–70°Cbeforefixing.Ifslidesarestoredat–70°C,warmtheslidestoroomtemperaturebeforethefixingstep.

5. Placetheslidesin100%acetoneat4°Cfor10minutestofixthesections.Removetheslidesfromacetoneandairdryfor10–30minutes.

6. CircleeachtissuesectionusingtheMiniPAPPen.Storeat–70°Cuntilreadytouse.

7. TheslidesshouldbewashedinPBSfor10minutespriortostainingormounting.

Paraffin-embedded sections—deparaffinization and rehydration Tousetheformalin-fixed,paraffin-embeddedsectionsforimmunohistochemicalstaining,deparaffinizeslideswithxyleneandthenrehydratein

agradedseriesofalcohol.

1. Topreparesections,dryslidescontaining4µmformalin-fixed,paraffin-embeddedsectionsina55°Covenfor2hoursorovernight(donot

allowthetemperaturetoexceed60°C)andstoretheslidesatroomtemperatureuntilneeded.

2. Placetheslidesinxylenefor5minutesatroomtemperaturetodeparafinize.

3. Removeslidesandplaceinasecondxylenewashfor5minutes.

4. Removetheslidesandplaceintwosuccessive100%ethanolwashesfor5minuteseach.

5. Next,placetheslidesin95%ethanolfor5minutesandthentransferto80%ethanolfor5minutes.

6. Removetheslidesfromthe80%ethanolandplaceinPBSfor10minutes.

7. Drainanyexcessreagentbytappingtheedgeoftheslideonpapertowelsandwipetheareanearthetissuesectionswithalaboratorywipe.

8. CircleeachtissuesectionusingtheMiniPAPPen.Theslidesarenowreadytouse.

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Protein extraction from tissues

Sample preparationPropersamplepreparationiskeytothesuccessofawesternblotanalysisexperiment.Variousfactorsaffectthedesignofthesamplepreparation

protocol.Duetothelargevarietyofproteinspresentindifferentcellsandtissues,itisnotpossibletohaveasinglesamplepreparationprotocol

thatissuitableforallproteins.Basedonthestartingmaterialandgoaloftheexperiment,thesamplepreparationprotocolneedstobedeter-

minedempirically.Thesamplepreparationconditionsmayalsobeoptimizedbasedonyourinitialresults.Generalguidelinesareprovidedbelow

topreparesamplesfromvarioussources,andexampleproceduresareprovided.

Mammalian tissue samplesTheprotocolbelowdescribesthepreparationofamammaliantissuelysatefrom100mgtissueusing1mLCellExtractionBuffer.Thefollowing

protocolissuitableforusewithavarietyoftissuetypes,althoughsomeoptimizationmaybenecessaryforsometissuetypes.

1. Ifneeded,cutthetissueintosmallerpiecesandplace~100mgtissueinamicrocentrifugetube.

2. Add1mLCellExtractionBuffercontainingProteaseInhibitorCocktail.

3. Homogenizethetissueusingapestlethatfitssnuglyintothemicrocentrifugetube.

4. Incubatethesamplesonicefor10minuteswithintermittentvortexing.

5. Centrifugethelysateat10,000xgfor5–10minutestoremoveanyparticulatematerial.

6. Transferthesupernatanttosterilemicrocentrifugetubesandaliquotintosingleusetubes.

7. ProceedtoPreparingSamplesforSDS-PAGEafterproteinestimation,orstorealiquotsat–80°C.Formoredetailedinformation,followpro-

tocoldescribedinthekit(Cat.No.WB7401).

Preparing tissue for flow cytometry analysis1. Followingeuthanasia,collectthetissueofinterestandplaceitincoldPBS.

2. Mincethetissueinto~1mmcubesusingarazorbladeina35mmPetridishcontaining1–2mLofLiverDigestMedium(17703-034).

3. Incubate15to45minutes,dependingonthedegreeofconnectivetissue,andpipetteupanddownevery15minutesusinga5mLpipettor

tomix.

4. Followingincubation,add4–5mLofPBS,andusea100µmstrainertogentlyforcecellsthroughthestrainerintoa10mLconicaltube.

5. Washtheisolatedcellsbyadding5mLPBSandcentrifugingat1,000xgfor5minutes.

6. Discardthesupernatantandrepeat1–2xforatotalof2–3washes.

7. Ifthepelletisred,indicatingahighnumberofredbloodcells(RBCs),washthepelletwithErythrocytesLysisBuffer(L5)fromthePureLink™

TotalRNABloodKit(K1560-01).

8. IfyouneedtoremoveRBCs,add500µLofL5andincubatefor10minutesonice.

9. Gently vortex the tube 2–3 times during the incubation step to promote complete lysis of erythrocytes. The solution should become

translucent.

10. Centrifugethetubeina4°Ccentrifugeat400xgfor10minutes(tomakesurecelldebrisisnotretainedsupernatant).Carefullyremovethe

supernatantanddiscardit.

11. GentlyresuspendthecellpelletinPBSandproceedwiththewash.Thecellpelletshouldbewhitewithnotracesofred.

12. Resuspendthepelletinto0.5%paraformaldehydeandforcecellsthroughstrainer(50µm)beforeflowcytometryanalysis.

SPECIAL NOTE AND DISCLAIMER: Literature describing in vivo delivery of siRNA and modified siRNA has become more abundant. However, the applications and methods described often vary.

To the extent Invitrogen provides general guidelines for using siRNA in animals, the company makes no guarantees concerning use of these products or guidelines in animal studies.

In vivo RNAi5



FEATURED STUDY

Stealth RNAi™ siRNA in vivo delivery to mouse brain as a model of Alzheimer’s disease

Effective knockdown achieved in an Alzheimer’s disease model using Stealth RNAi™ siRNA. (A) Experimental design: A transgenic mouse model forAlzheimer’s(hAPPTg)overexpressingthehuAPPgenecarryingLondonandSwedishmutationsunderthemThy1promoterwasused.Acannulawasinsertedintothelateralventricleofthebrain.StealthRNAi™APPsiRNAsolution(4.75mg/mLinPBS)wasinfusedviacannulaat6μL/dayfor2weeksinthelateralventricleusinganosmoticminipump.(B)BrainhomogenatesfromStealthRNAi™siRNA–treatedmicewereseparatedintocytosolicandparticulatefractionsasdescribedinRockensteinetal.[3].Westernblotswereprobedwithamousemonoclonalanti-hAPPantibody,screened,andanalyzed.(C)Thefixedbrainswereseriallysectionedat40μm,andsectionswereimmunolabeledwithanti-hAPPantibody,followedbyincubationwithaFITC-labeledsecondaryantibody,andimagedwithaQuantimet™570Csystem(Leica)orbyLSCMaspreviouslydescribed.APPproteinlevelisreducedintheneocortextreatedwiththeStealthRNAi™APPsiRNAbutnotwhentreatedwiththeStealthRNAi™siRNAControl.

mThy-1 gene

APP mut

APP

Injection

Intracerebral

Stealth RNAi™ siRNA

Hippocampus

Cortex

120

Non-transgenic

Stealth RNAi™ siRNA Control

Stealth RNAi™ APP siRNA

42

hAPP

Actin

hAPP transgenic

Inte

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leve

ls

NontransgenicStealth RNAi™

APP siRNA Stealth RNAi™

siRNA Control

2,000

0

4,000

6,000

8,000hAPP levels

hAPP transgenic

Nontransgenic

hAPP transgenic

0

100

200

300

Inte

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leve

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hAPP levels

Stealth RNAi™

APP siRNA Stealth RNAi™

siRNA Control

hAPP

Stealth RNAi™ siRNA control Stealth RNAi™ APP siRNA

A

B

C

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Frequently asked questions about in vivo RNAi

Looking for an answer to your question? Browse the following topics for an answer. For answers to additional questions, please refer to the

Invitrogen’stechnicalsupportFAQdatabaseorcontactInvitrogenTechnicalSupporttohavearepresentativeassistyou.

Q. Should I use chemically modified duplexes for my in vivo RNAi experiments?A.ChemicallymodifiedStealthRNAi™siRNAduplexeshaveanumberofadvantagesoverstandardRNAiduplexes,includingminimizationofoff-

targeteffects,enhancedstability,andreducedtoxicity.Forthesereasons,StealthRNAi™chemicallymodifiedRNAiduplexesarerecommended

forin vivo RNAiexperiments.Forsomepilotexperiments,wherethegoalistodeterminebiodistribution,siRNAduplexesandfluorescentcontrols

areusefulandmorecost-effective.

Q. Should I use labeled or unlabeled duplexes for my in vivo RNAi experiments?A.WehavedemonstratedthatlabelingStealthRNAi™siRNAduplexesdoesnothampertheirknockdownpotency.Analternativeapproachisto

mixunlabeledduplexeswithlabeledcontrolduplexes;thismethodismorecommonlyusedwithin vivo RNAi,andeliminatespossibleeffectsof

thedyeonthetarget,whichisusefulshouldyouwishtousethedataasamodelforfutureclinicalstudies.

Q. I am planning on using siRNA for my in vivo experiments. What purity should they be?A.Invitrogen’sproductionofin vivoRNAiduplexesbeginswithstandardsynthesisofRNAioligosusinghigh-qualitystartingmaterials.TheRNA

oligosarethenduplexedanddesalted.Atthispoint,youcanalsorequestHPLCpurification,priortofurtherin vivopurityprocessing.However,this

stepincreasescostandreducesyield.Subsequentin vivopurityprocessingincludesaseriesofdialysisandcounterionexchangestepstoremove

toxicsaltsandsolventsfromtheRNAduplexandtolowertheconductivitytophysiologicalconditions.Theresultinghigh-qualityduplexesare

readyfor in vivo useregardlessofwhetherHPLCpurificationisrequestedupstreamofthisprocess.

Q. Should I use siRNA or vectors for in vivo RNAi?A.RNAicanbedeliveredusingtwodifferentapproaches:siRNAsyntheticduplexesorsiRNAexpressedfromplasmidsorviralvectors(shRNA,

miRNAi).siRNAsarethemethodofchoicefortherapiddevelopmentoftherapeutics.Theyareeasydesign,easytosynthesizeandeasytouse.siR-

NAscanberapidlyidentifiedandmultiplegenescanbetargetedatthesametime.WithRNAivectors,theexpressionwillbesteadierasaresultof

thepotentialfortheplasmidtostablyintegrateintothegenome.Additionally,RNAivectorsareabletotargetnondividingcellssuchasstemcells,

lymphocytesandneurons.Thedrawbacks include thedangerofoncogenic transformation from insertionalmutagenesis, andunanticipated

toxicityfromlong-termsilencingofhumangenesand/ortheexpressionofhighamountsofsiRNAinsidethecell[1].

Q. How should I deliver my in vivo RNAi molecules?A.SeveraldifferentapproacheshavebeenusedforsiRNAdelivery,includingvariouslocaldeliverytechniquesandsystemicdelivery.

Q. How many micrograms or milligrams are delivered of the nanomole quantities of synthetic duplexes offered by Invitrogen for in vivo RNAi? A.Table5.4providesnanomolequantitiesinmicrograms(µg)andmilligrams(mg).

Table 5.4. Quantities delivered, in micrograms (µg) and milligrams (mg).

siRNA Stealth RNAi™ siRNA

Purity Quantity µmol µg mg µg mg

HPLC5nmol

20nmol500nmol

0.0050.020.5

67269

6,731

0.0670.2696.731

81322

8,050

0.0810.3228.050

Desalt—in vivopurity

25nmol100 nmol

2µmol

0.0250.12

3371,346

26,922

0.3371.346

26.922

4031,610

32,200

0.4031.610

32.200

HPLC—in vivopurity

5nmol20nmol

500nmol

0.0050.020.5

67269

6,731

0.0670.2696.731

81322

8,050

0.0810.3228.050

In vivo RNAi5



Q. I want to order larger scales than the standard offering. Whom do I contact?A.Pleasecontactrnairesearcher@invitrogen.comregardinglarge-scalein vivoRNAi

Q. Do I need endotoxin testing for my in vivo RNAi duplexes?A.Pleasecheckwiththeappropriateregulatoryagencyforguidance.

Q. How can I order in vivo RNAi duplexes?A.RNAiduplexesforin vivoexperimentscanbeorderedusing thesimpleBLOCK-iT™RNAiExpress.

Q. How much is the price per duplex of the in vivo RNAi duplex I wish to purchase?A.Pricingiscalculatedinthefollowingmanner:

Duplex price = (purity price) + (price per base for chosen scale x quantity of bases) + (optional 5’ sense strand label) + (optional endotoxin testing)NOTE:siRNAhas42bases.StealthRNAi™siRNAhas50bases.

Chapter references1. GrimmDetal.(2006)Nature441:537–541.

2. LayzerJMetal.(2006)Biochem Biophys Res Commun344:406–415.

3. RockensteinEetal.(2001)J Neural Sci Res66:573–558.

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Chapter 6siRNA screening

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CHAPTER 6

siRNA screening

Selecting an siRNA library

Customer insights: what is important when selecting an siRNA library?Datageneratedfromalarge-scalesiRNAscreeningexperimentareinvaluablewhensearchingforgenetargetsinvolvedinaparticularbiologi-

calprocess,pathway,ornetwork.However,obtainingalarge-scalesiRNAlibraryandpreparingtorunthefirstscreencaninvolveasignificant

investment.Manyfactorsshouldbeconsideredpriortoinitiallibrarypurchase,includingthequalityofthereagents,formattingneeds,validation

strategy,andsupport.Recently,weinterviewedscientistsfromthetopsiRNAscreeninglaboratoriesandhereweprovideasummaryofthefactors

theyrecommendyoushouldconsiderbeforepurchasingansiRNAlibrary.

Key consideration: siRNA design and performanceLarge-scalesiRNAscreenscangeneratehundredsofpotential“hits”—genesforwhichoneormoresiRNAsgiveadesiredphenotype.Unfortunately,

inearlyscreenswithunmodifiedsiRNAtechnologiesandpooledsiRNAapproaches,manyofthesehitsactuallyresultfromoff-targeteffects.Off-

targeteffectscanresult frompoorsiRNAdesign,usingansiRNAtechnologythat isnotdesignedtoreduceoff-targeteffects,orusingsiRNAs

withlimitedpotency,whichrequiresdeliveryofalargeamountofsiRNAtoobservegenetargetknockdown.“Thefirstconcernisthequalityof

thealgorithmthatisusedtodesignthesiRNAs,”saysAnandGanesan,MD,PhD,AssistantProfessor,DepartmentofDermatologyandBiological

ChemistryattheUniversityofCalifornia,Irvine.Secondly,thenumberofsiRNAsthathavebeenvalidatedeitherbythecompanyorasapartof

otherstudies”shouldbeconsidered.Ambion®Silencer®SelectsiRNAsincorporatethelatestimprovementsinsiRNAdesign,off-targeteffectpre-

dictionalgorithms,andchemistry.Asaresult,theyenableunrivaledsilencingconsistency,potency,andspecificity,andfewerfailedexperiments,

allowingcleaner,moreconsistentphenotypicdata(Figures6.1and6.2).

Silencer®SelectsiRNAsaredesignedwithanewlydevelopedalgorithmandincludelockednucleicacid(LNA®)chemicalmodifications,mak-

ingthemthebest-performingsiRNAscommerciallyavailable.Experimentsdesignedtoassesslevelsofknockdownandobservedphenotypes

showthatLNA®-modifiedSilencer®SelectsiRNAsconsistentlydemonstratethehighestlevelsofknockdownandtheleastoff-targeteffects[1].To

date,morethan4,300Silencer®SelectsiRNAstargetedtothemostpopulargenetargetshavebeenbenchtestedandvalidatedataconcentration

of5nMtoprovide≥80%targetmRNAknockdownasmeasuredbyqRT-PCRwithTaqMan®GeneExpressionAssays.Thisextensivelevelofvalida-

tionprovidesenhancedconfidenceinboththeSilencer®SelectsiRNAdesignalgorithmandthesiRNAlibrary’soverallperformance.

Figure 6.1. Silencer® Select siRNAs provide up to 100x higher potency com-pared to other siRNAs. Silencer®SelectsiRNAsto10differenttargetsandsiRNAsfromtwoothersupplierstothesame10differenttargetswereindividually transfected into HeLa cells in triplicate at the indicatedsiRNAconcentrations.mRNAknockdownlevelsweretested48hrlaterasdescribedinFigure6.2.AveragepercentmRNAremainingisshownforeachsetofsiRNAs.

Ave

rage

% m

RN

A r

emai

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100

80

60

40

20

00.03 nM 0.3 nM 3 nM 30 nM

80% Knockdown

siRNA concentration

D modi�ed (n = 40)Q unmodi�ed (n = 30)Silencer ® Select (n = 30)

Figure 6.2. Silencer® Select siRNAs elicit expected phenotype at a higher rate than other siRNAs. siRNAsto7genetargetswithwell-understoodRNAi-inducedphenotypeswereindividuallytransfectedat3nMandphenotypesmeasured48hrlater.EachbarrepresentsthepercentofsiRNAsthatgavetheexpectedsilencedphenotype.siRNAstoBUB1B,AURKB, WEE1, and PLK1 were assessed using a multiparametric cellgrowth/apoptosisassayinU2OShumanosteosarcomacells.siRNAstoHMGCR,LDLR,andFDFT1wereassessedusinganLDLuptakeassayinHUH7humanhepatomacells.

% s

iRN

As

with

exp

ecte

d p

heno

typ

e

100

80

60

40

20

0A

modi�edn = 28

B unmodi�ed

n = 21

Silencer ® siRNAn = 34

Silencer ® Select siRNAn = 43

siRNA screening6



Key consideration: siRNA qualitysiRNA design and structure is important; however, siRNA manufacturing quality is equally critical. Dr. Hakim Djaballah, Director of the High

ThroughputScreeningCoreFacilityatMemorialSloanKetteringCancerCenter,said,“Cellularviabilityisamajorconcerninourscreeningexperi-

ments.WeneedtoknowthatthecontaminantsthatresultfromtheproductionofsiRNAareatextremelylowlevels.Oftenwediluteourreagents

andusethemonasmallscalewithasmallnumberofcells,andwecannotaffordimpuritiestobepresent.”Ambion®Silencer®SelectsiRNAsare

manufacturedunderstringentcontrolandaresubjectedtorigorousqualitycontrolprocedurestoensurelot-to-lotconsistency.Eachandevery

strandissubjectedtoMALDI-TOFmassspec,andeachannealedstrandistestedbynondenaturingPAGEtoensureefficientannealing.Theaver-

agepurityof“standardpurity”siRNAstrandsisapproximately90%full-lengthproduct.

Key consideration: completeness of the libraryAllscreeningscientistsagreethatacompletesiRNAlibraryiscrucialtosuccess.ThelibraryshouldcontainsiRNAstoasmanyrelevantgenesaspossible,

andmostresearchersprefertoerronthesideofscreeningtoomany,ratherthantoofewtargets.TheAmbion®Silencer®SelectsiRNAalgorithmhasbeen

usedtogeneratesiRNAstothehuman,rat,andmousegenomes,andansiRNAlibraryfor21,585uniquegeneswithinthehumangenomeisavailable

forimmediateshipment.Otherspeciesarecoveredonacustombasis.PredesignedsiRNAssetsareavailableforanumberofimportanthumangene

familiesthat includekinases,phosphatases,GPCRs,andseveralothers.Anextendeddruggablegenomeset isavailablethattargets10,415genesof

potentialtherapeuticinterest.Althoughpreparedsetsareveryuseful,moreandmoreresearchersprefertoscreentheirownsubsetsoftargets.Tothat

end,AmbioncanprovideacustompreparationofSilencer®SelectsiRNAstargetingyourlistoffavoritegenes.Finally,ourbioinformaticsteamisstanding

bytohelpyougeneratealistofgeneswithinafunctionalclassorbiologicalpathwayforyourcustomsiRNAlibrary.

Key consideration: customizationsiRNAscreensarequitevariedinnature.“Everyexperimenthasdifferentrequirements.WhatisthescaleofsiRNAdesiredineachwell?Howare

theduplexesoriented?Canthesupplierprovidecustomaliquotting?Andmostimportantly,whataretheturnaroundtimesfororderedlibraries?”

saidDr.Djaballah.AllarecriticalfactorsinselectingansiRNAlibraryvendor,bothfortheinitialsiRNAlibrarypurchaseandforhitconfirmation

follow-upstudies.

Ambion®Silencer®SelectsiRNAlibrariesareavailableatscalesof0.1,0.25,1.0,2.0,and5.0nmolperwell.Customaliquottingrequestsofall

typesareeasilyhandled,andwecansupplysiRNAsinyourdesiredplatelayoutstomatchyourtransfectionandcell-basedassayprotocols.

Key consideration: validationOncehitsfromalibraryscreenareidentified,validationisacrucialsteppriortodrawingconclusions.Typically,hitsarevalidatedbyrepeatingthe

experimentwiththesamesiRNAsequences,usingasecondaryassayoradditionalsiRNAsequences,orusingqRT-PCR,oftenfollowedbylooking

atproteinlevelstoassesstargetknockdown.“Wedon’tthinksimplyusinganothersupplier’ssiRNAisagoodenoughmethodforvalidationif

youarerelyingonthedifferencesbetweenthetwosuppliers’algorithms.WepreferqRT-PCRfollowedbyproteinlevelmeasurements.Wesome-

timeslookatrescueexperimentsusinganexpressionvector,butthesecanbetediousandtherecanbetoxicityconcerns,”saidDr.Djaballah.Dr.

Ganesanremarked,“Wevalidatehitsbyseveraldifferentstrategies.WeutilizemultiplesiRNAstoeliminateofftargets.WedoquantitativeqRT-PCR

toensurethatthesiRNAimpactstheexpressionofthetargetgene,andweexaminetheimpactofthesesiRNAsinseveraldifferentcelllines.More

recently,wehavealsobeenvalidatingourhitswithshRNAs.”Dr.Ganesanwentontosay,“Themajorcostofscreensisusuallynotfromthescreen

itself,butfromthefollow-ups.”Keystosuccessfulvalidationarehavingadefinedstrategyupfrontandhavingastreamlinedmethodforobtain-

ingadditionalsiRNAsforvalidationpurposes.Wecanhelpyoudefineanappropriatefollow-upandhitvalidationstrategy.Since2002,Ambion

hasconsultedwithandhelpedthousandsofscientistsusingsiRNAs.CustomsetsofSilencer®SelectsiRNAsareavailablewithaslittleas0.1nmol

siRNAperwell—arrangedinplatesanywayyoulike.Ofcourse,followinguponlargenumbersoffalsepositiveresultscaneasilydrainyourhit

validationbudget.ByperformingourprimaryscreenwithSilencer®SelectsiRNAs,whichreduceoff-targeteffectsbyupto90%,andbypurchasing

thelowestamountofsiRNAyouneed,youcankeepfollow-upsiRNAcoststoaminimum.

Key consideration: communication and supportWerealizethatpurchasingtheinitialsiRNAlibrary,settingup,performing,andvalidatingansiRNAscreencanbealargeendeavor.Dr.Djaballah,

whohasdirectedmanysiRNAscreeningexperiments,statedthat“theremustbeaccesstoopendiscussion”whenitcomestoplanningascreen.

Wecertainlyunderstandthisneed.Tothatend,weencourageopencommunicationthroughsiRNAlibraryusergroupmeetings,wesupport

majorRNAisymposia,andofferfreeconsultationsonyoursiRNAscreeningprojecteverystepoftheway.Ourscientistsandaward-winningtech-

nicalsupportstaffwelcometheopportunitytosupportyouinyoursiRNAexperiments.

For more information about Ambion® Silencer® Select siRNA libraries, please contact your local Invitrogen sales representative or visit

www.invitrogen.com/rnai.

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DEFINITIONS

siRNA librariesSetsofsiRNAs,usuallyprovidedindividuallyin96-wellor384-wellplates.siRNAlibrariesmaytargetanynumberofgenes,e.g.,anentire

genome,agenefamily,abiologicalpathway,oracustomsetofgenes.

HitsForthisapplicationguide,a“hit”isdefinedasagenethattriggersthephenotypebeingassayedinaninitialsiRNAlibraryscreenwhenit

issuccessfullytargetedbyansiRNA.Inotherwords,hits(alsocalledcandidateorputativehits),arepositiveresultsfromansiRNAlibrary

screen—theymayresultinaphenotypethatissimilartothepositivecontrolinthescreeningassay.Ideally,candidatehitsrepresentgenes

thatdirectlyorindirectlyinfluencethecellularprocessunderstudy.Confirmedhitsarethosethathavebeenvalidatedusing2–3methods

tocorroboratetheinitialscreeningresults.

NegativesGenesforwhichnoneofthe targetingsiRNAsresultsinahit.

False positivessiRNAsthatgiveapositiveresult inthescreeningassaythatarenotconfirmedbyotherdistinctsiRNAstargetingthesamegeneorby

secondaryassays.Theanomalousassayresultfromafalsepositivemaybeduetooff-targeteffectsofthesiRNAortoexperimentalerror.

siRNAscreensshouldbedesignedtoruleoutallfalsepositives.

False negativesTruehitsthatarenotdetected.ThesesiRNAsgiveanegativesignal;however,useofotherdistinctsiRNAstargetingthesamegeneshow

thatwhenthistargetissilenceditgivesapositiveassayresult.Falsenegativeresultscancauseyoutooverlooktruepositivesandshould

beminimized.

Biological false negativesOccurwhentheRNAireagentscannotsilenceatargetsufficientlytorenderitrate-limiting—thus,nophenotypeisobserved.Forexample,

thetargetproteinmayhavealonghalf-life,orextremelyhighactivity,sothatverylittleproteinisneededtofulfillitsfunction.Theremay

alsoberedundancyoffunctioninthepathway,suchthatthefunctionofthesilencedgeneproductisreplacedbytheproductofasecond

gene.Thesetypesoffalsenegativesaredifficultifnotimpossibletoavoid.

Technical false negativesArecausedbysuboptimalscreeningreagents,conditions,orassaydesign.Theycanalsoresult fromexperimentalvariability.While,of

course,onewouldliketodetectasmanypotentialhitsaspossible,allowingforahigherrateoftechnicalfalsenegativesmaybeworththe

significantdecreaseinoverallcostsandtimerequiredtoperformthescreen.

Screening with siRNA libraries

AdvantagesRNAiiscurrentlytheeasiestandmostcost-effectivereversegeneticstoolforstudyinggenefunction.Plate-basedandcell-basedassays,including

highercontentmultiparameterassaysandtheavailabilityoflibrariesofeffectivesiRNAs,allowresearcherstoquicklyevaluatedozenstohundreds

ofgenesfortheirroleincellularprocesses.siRNAlibrariescanbeobtainedinavarietyofformats,includingsingle-transfection,ready-to-usever-

sions,makinggenome-wideorsmallerRNAiscreenspossibleinalmostanylaboratory.

siRNA screening6



ChallengesWhilemanylaboratoriesareperformingRNAiscreenswithsiRNAlibrariestodeterminegenefunctioninmammalianculturedcells,thetechnol-

ogy is justnowbeingbroadlyapplied. Inthepast, threefactorshave limitedtheadoptionofthistechnology: (1)theperceivedcostofsiRNA

libraries,(2)thebeliefthatexpensiveroboticliquidhandlingsystemsarerequired,and(3)theanticipateddifficultiesinperformingtransfections

reproduciblyin96-or384-wellplates.

Toolsarenowavailablethataddressalloftheseissues,makingsiRNAlibraryscreeningfeasibleformorelabsthaneverbefore.siRNAlibraries

areavailableinformatssuitableforasingletransfection,anduptothousandsoftransfections.Therearenowextensivesetsofpositiveandnega-

tivecontrolsanddeliveryoptimizationreagents fordevelopingoptimalscreeningprotocols.High-throughputdeliveryreagents, instruments,

andprotocolsyieldeasyandreproduciblesiRNAdeliveryinmultiwellplatesusingeithermanualorroboticmeans.

KeepinmindthatRNAifacilitatesgeneknockdown,notgeneknockout;i.e.,targetgeneexpressionisalmostnevercompletelyextinguished.

Whether the level of knockdown is sufficient to generate a positive outcome in the screening assay (rendering expression rate-limiting) will

dependontheendogenousexpressionlevelofthegene,ontheproteinproduct’sactivityandhalf-lifewithinthecell,andonredundancyof

functionwithinthatbiologicalpathway.Someproteinsmayrequireverylittleactivitytoaffectabiologicalprocess,andare,ingeneralendog-

enouslyexpressedatmuchhigherlevelsthanbiologicallynecessary.Thus,theirresidualactivityafterRNAiknockdowncouldstillbesufficient

tofulfilltheircellularrole.Itmightthereforebedifficult,forexample,toachievesufficientgeneknockdowntoextinguishtheeffectsofcertain

highlyactiveproteins.

GoalsDesignthesiRNAscreen forcomprehensivecoverageof thegenes involved in thephenomenonbeingstudiedwhileminimizingthe rateof

technicalfalsenegatives(biologicalfalsenegativescannotbeavoided;seeDefinitionsonpage31).Bycarefuldesignofexperimentalcontrolsand

assessmentofresults,thescreenshouldalsostrivetoeliminatefalsepositives.

Thesegoalsarebestattainedthroughsequentialscreeningpasses,ideallyusingindependentassaysforeachasfalsepositives.Theinitialpassis

focusedonmaximizingsensitivityofdetection,andthereforeshoulduseconditionsthatfavormaximalsilencing(e.g.,usemultipleindividualsiRNAsper

targetatrelativelyhighconcentrations(30–100nM)).Dependingonthetypeofassayandthethresholdused(usually2–3standarddeviationsfromthe

baseline),arelativelyhighpercentageoftargetedgenesmayshowapositivephenotypefromatleastonesiRNA,warrantingfollow-upevaluation[2].

Thesecandidatehitswill,nodoubt,includenumerousfalsepositives,whichwillbefilteredoutusingvalidationexperiments.

siRNA library screening workflow (5 steps)

RNAiscreeningusingansiRNAlibraryinvolvesthefollowingfivesteps;theyarebrieflyintroducedhereandaredescribedindetailinlaterchapters.

→ Step1.Experimentaldesign

→ Step2.OptimizethesiRNAdeliverymethod

→ Step3.Developandoptimizeaneffectivescreeningassay(s)

→ Step4.Performscreenandanalyzedata

→ Step5.Validatescreeningresults

Step 1. Experimental designAmongthemostcriticalaspectsofexperimentaldesignforsiRNAscreeningareselectionofsiRNAstoscreenandtouseascontrols, thecell

type(s)inwhichtheexperimentistobeperformed,andthesiRNAdeliverystrategy.Thesedecisionsshouldbebasedonthebiologicalpathway

understudy.Forinstance,tolookatoncogenesis,youmaywishtoscreenansiRNAlibraryfordifferentialeffectsoncellproliferationinacancer

cell linecompared toanoncancerouscell line.Thechoiceof siRNA librarywilldependon thedepthof thescreenand theoveralldesignof

theexperiment.Doyouwanttoanalyzeeverypossibletarget,orjustpotentiallydruggabletargets,suchaskinases?siRNAlibrariesthattarget

completegenomes(human,mouse,etc.),aswellassetsofsiRNAsthattargetspecificgenefamiliesorbiologicalpathwaysareavailable.Libraries

canalsobecustomdesigned;thepossibilitiesarealmostendless—itisuptoeachindividualresearchertodeterminehowbesttoapproachthe

experimentalproblemathand.

Equally importantinchoosinganappropriatesetofsiRNAsforyourscreen, istousewell-designed,efficacioussiRNAswithahighprob-

abilityofinducingsuccessfulknockdown.EffectivesiRNAdesigniskeytoavoidwastingbothtimeandmoney.Weuse“intelligent”siRNAdesign

algorithmsandvalidateaselectionofoursiRNAs.Ultimatelythemostimportantattributeofanyalgorithmisitsperformancewhentestedexperi-

mentally.ThereforeanalgorithmthathasbeenprovenusingmanysiRNAstargetingmanydifferentendogenoustranscriptswillproducemore

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effectivelibrariesleadingtomoreusefuldata.Additionally,algorithmsthathavebeentrainedtopredictthehighestpotencysiRNAswillallowyou

tousesiRNAsinyourexperimentsatlowerconcentrations,resultinginmoreeffectivescreeningperformance.

EvenwithextensivevalidationofansiRNAdesignalgorithm,thevastmajorityofsiRNAsinalmostallsiRNAlibrarieshavenotbeenexperi-

mentallyproventoknockdowntheirintendedtarget.TheuseofmultiplesiRNAspertargetisgenerallyacceptedtobethebestapproach[3].

Employing three or more distinct, individual siRNAs, and performing replicate transfections, provides statistical significance and significantly

decreasesbothfalsepositiveandfalsenegativeratesascomparedtoscreeningwithpoolsofsiRNAs.

Another important part of the process of selecting siRNAs for screening is to choose appropriate positive and negative control siRNAs.

PositivecontrolsiRNAsarenecessarytoprovethatthesiRNAsinthescreenaredeliveredefficientlyandcaninducesilencingintheexperimental

conditionsused.Additionalassay-specificpositivecontrolsiRNAsarenecessarytoensurethatthescreeningassayyieldssufficientsignalforaccu-

ratequantitationofthedata.Nontargeting,negativecontrolsiRNAsarenecessarytocontrolfornonspecificeffectsduetosiRNAdeliveryandto

screenassaybackground.Finally,inscreensthatincludeatreatmentofsomesorttoinducethephenotypiceffect,untreatedcontrolsserveasa

baselineforthescreeningassay.

Step 2. Optimize the siRNA delivery methodScreeningsiRNAs that targethundreds to thousandsofgenes requiresconditions forhigh-throughput siRNAdelivery. Furthermore,efficient,

reproduciblesiRNAdeliveryiscriticalforeffectivesiRNAlibraryscreens.UsingoptimalsiRNAdeliveryconditionseliminatesthemostcommon

causesoffailedgenesilencingexperiments.Becauseitissoimportant,wehighlyrecommendinvestingthetimetoselectthebestsiRNAdelivery

methodandconditionsforthecellsthataretobeusedforsiRNAlibraryscreening.

TherearetwobasicmethodsemployedforsiRNAdelivery:lipid-mediatedtransfectionandelectroporation.Lipid-mediatedreversetrans-

fectioncanbeusedtotransfectupto1,000wells in lessthananhourwithouttheneedforrobotics[4].Withautomatedliquidhandling,the

pacecanbeevenfaster.ElectroporationcanalsobeusedtodeliverdozenstohundredsofsiRNAsquicklywhenperformedwitha96-wellplate

electroporationchamber.

The first step in optimizing siRNA delivery conditions for an RNAi screen is to identify a transfection reagent and plating conditions, or

electroporationconditions,thatmaximizeuptakeofactivesiRNAwhilemaintaininghighcellviability.WefinditusefultomeasurebothsiRNA-

inducedtargetknockdownandcellviabilityincellstransfectedwith2–5differenttransfectionreagentsor5–10electroporationconditions(e.g.,

varyingcellconcentration,voltage,pulselength,pulsenumber,siRNAconcentration,etc.).Deliverycanthenbefurtheroptimizedforthereagent

orelectroporationconditionthatworkedbestamongtheconditionstested.Choiceofdeliverymethodanditsoptimizationwilldependonthe

celltypeused.

Step 3. Develop and optimize an effective screening assayTheresultsofsiRNAlibraryscreensaretypicallyevaluatedusingphenotypicassays,suchasreportergeneassays,cell-basedassays,plate-based

assays,etc.ratherthanthroughdirectmonitoringofchangesintargetmRNAorproteinlevels.ThesuccessofansiRNAlibraryscreendepends

greatlyonthequalityofthephenotypicassayusedforscreening.Thus,itisworthwhiletoinvestthetimeandeffortrequiredtocreateanassay

withenoughprecision,signal-to-noise,andlinearrangetoensureidentificationofsiRNAsthatinducethedesiredphenotypeintransfectedcells.

ThediversityofcellfunctionsthatcanbecharacterizedusingsiRNAlibrariesislimitedonlybytherangeofphenotypicassaysthatcanbe

developed.AssaysthatareamenabletosiRNAscreeningexperimentsrangefrommicroscopicassaysthatmonitorcellsize,cellcyclestatus,or

antibodystaining;toenzymaticassaysthatassesstheturnoverofaspecificsubstrateinacelllysate;todirectmeasurementsofbiomoleculesor

smallmoleculesinlysates,oncells,orinmedium.

Goodquantitativeassayswilldemonstrateahighsignal-to-noiseratio;thatis,theywillexhibitalargedifferenceintheresultsobtainedfrom

assay-specificpositivecontrolsandnontargetingnegativecontrols(whichmayserveasnegativecontrolsforboththephenotypicassayandfor

silencing).SuchassayswillyieldawiderangeofresultsfromsiRNAscreeningexperiments,andtheywillmaximizethechanceofidentifyinggenes

withaninterestingphenotypewhensilenced.Maximizingthesignal-to-noiseratioinvolvestestingvariableslikeassaytime,assaycomponents

(e.g.,thereporter),celltype,andlengthoftimebetweentransfectionandassay.

Step 4. Perform screen and analyze dataWitharobustphenotypicassayandoptimizedsiRNAdeliveryconditionsestablished,alibraryofindividualsiRNAscanbeintroducedintocells.

TriplicatetransfectionsforeachsiRNAprovideenoughdataforreasonablestatisticalanalysis.UseofthreesiRNAspertargethelpstoeliminate

nonspecificeffectscausedbyanysinglesiRNAsequence(falsepositives)aswellasanyfalsenegatives.Positivecontrolandnegativecontrols

siRNAsoneachplateprovidequantitativenumberstonormalizedatafromdifferentplates,andtoprovidetherangeofphenotypesthatcanbe

usedtoidentifysiRNAsthatyieldapositiveeffect,designated“candidatehits”(seeDefinitionsonpage31)Thedataarecollectedandanalyzed

toidentifycandidatehits.Inmanycases,werecommendreservingthisdesignationfortargetgenesthatexhibittheexpectedphenotypewhen

siRNA screening6



transfectedwithtwoofthethreeindividualsiRNAs.Thisshouldeliminatefalsepositivesandfalsenegatives[5].Anotherapproachistofollowup

onallofthesiRNAstoagivengeneevenwhenoneonegaveapositiveresult.Thisapproachrequiresadditionalfollowupandfewpotentialhits

areconfirmed;however,itisagoodapproachtoavoidmissingtargets.

Candidatehitsareessentiallyaseriesofgenesidentifiedbytheinitialscreentobepotentiallyinterestingandthatwarrantfurtherstudy.Theyare

typicallyconfirmedusinganindependentmethodtoeliminate“falsepositives”andtoensurethatonlylegitimategenesareevaluatedfurther(Step5).

Step 5: Validate screening resultsTargetsidentifiedascandidatehitsintheintitalscreenmustbevalidated.Thisisdonebyasecondandoften,athirdscreeningpass.Theseexperi-

mentswillalsotypicallyshedlightontherolesofthetargetgenesinspecificbiologicalpathways.Anobviousvalidationtechniqueusedinsec-

ondaryscreensistoshowadirectlinkbetweentheobservedphenotypeandtargetgenesilencingbydocumentingareductionintargetmRNA

orproteinlevel.However,thespecificgoalsofthelibraryscreeningandtheassaytoolsavailablewilldictatethetechniquesusedforvalidation.A

majorgoalofthesecondscreeningpassistoretestallcandiategenestoidentifyinterestinghitsandeliminatefalsepositives.Sincefewergenes

areanalyzed,thesevalidationexperimentsmayalsoservetofurtherrefinetherelevanceofcandidatehitswithrespecttothebiologicalprocess

ofinerest.Itwouldnotbeuncommonforthesecondscreentoeliminateupto90%ofcandiatehitsidentifiedinthefirstpass.

Thosegenesconfirmedaspositiveinthesecondscreeningpasscanbesubjectedtoafinalthirdpasswherein,ifnotshownbyprevious

experiments, the functional phenotypic assay is repeated in parallel with a driect measure of target gene silcencing (usually by qRT-PCR or

branchedDNAassays),thusdirectlylinkingthetwo.Targetsthathavebeenvalidatedbytwoormorescreens,inaddition,totheinitialscreen-

ingexperiment,areexcellentcandidatesforfurtherstudies(e.g.,in vivostudies)designedtohelpindependentlyconfirmdataandexpandupon

resultsfoundinthepublishedliterature.

Toxicity

WhensiRNAsareintroducedintocellsviatransfection,unexpectedand/ortoxiceffectsmightbeobservedinadditiontotheexpectedpheno-

typesinvolvedinthescreen.

Off-targeteffectsareanygenesilencingeffectscausedbysiRNAsonnontargetmRNAsthroughtheRNAimechanism.Theycouldcomefromthe

guide(antisense)orpassenger(sense)strandofsiRNA.Inthecaseofnear-completecomplementaritythestrandswillinducecleavageofmRNA(actas

siRNA),whilethecaseoflimitedhomologycausetranslationinhibition(actasmiRNA).TheSVMalgorithmusedtodesignoursiRNAsselectsonlythe

sequencesthathavenoorlittlehomology(<15nt)tothetranscriptomeoutsidetheintendedmRNAtarget.Further,thepassengerstrandofsiRNAisinac-

tivatedbychemicalmodifications.However,ahighproportionof‘‘off-target’’transcriptssilencedbysiRNAshavebeenshowntohave3’-UTRsequence

complementaritytotheseedregionofthesiRNA,indicatingtheoff-targeteffectismediatedthroughanmiRNApathway.Sinceitisimpossibletoelimi-

nateall7–8-basematchesofsiRNAstothetranscriptome,itisdifficulttoachieveabsolutespecificity,evenwhenchemicalmodificationsareintroduced.

PerformingRNAiexperimentswithhyper-potentsiRNAsatlowconcentrations(<5nM)minimizesoff-targeteffects.

Anotherproblemcanarisefromcellularresponsestointroductionoftheforeignnucleicacid.Bothnon-immuneandimmunecellsmaybe

activatedbylongdsRNA,leadingtotheactivationofcytoplasmicreceptorssuchasthedsRNA-dependentproteinkinaseR(PKR)andtheretinoic

acidinduciblegene-I(RIG-I).OncePKRisactivated,itphosphorylatestheeukaryotictranslationinitiationfactor(EIF-2)-α,leadingtoglobalsup-

pressionofproteinsynthesisandsubsequentprogrammedcelldeath.PKRcanalsoactivatenuclearfactorκB(NF-κB)withconsequentinduction

oftype-IIFNproduction.Afamilyof2959-oligoadenylatesynthetases(2959-OAS)canalsobeactivatedbydsRNA.Thisleadstotheactivationof

RNaseL,whicheventuallytriggersthenonspecificdegradationofmRNA.

RIG-IisanintracellulardsRNAsensorcapableoftriggeringIFNproduction.TheRIG-Ihelicaserecognizesblunt-endedsiRNAs,leadingtotheactiva-

tionofdsRNAsignaling.RIG-IefficientlyunwindssiRNAscontainingbluntendsandtheefficiencyinduplexunwindingistranslatedintodownstream

signallingtointerferonregulatoryfactor3(IRF-3)andNF-κBactivation.Therefore,introductionoflongdouble-strandedRNA(>30bp),especiallyblunt-

endedsiRNA,caninducesevereantiviralresponsesintransfectedcells.Forthesereasons,typicalsyntheticsiRNAsareshorter,19bpinlength,andcontain

2ntoverhangsat3’-endstoreducecellularresponses.Inmostcasescellularresponsestosuchmoleculesareminimal.

MammalianimmunecellsalsoexpressafamilyofToll-likereceptors(TLRs),whichrecognizepathogen-associatedmolecularpatternsinclud-

ingCpGmotifsandviraldsRNA.Forexample,TLR7andTLR8were initiallyshowntomediate the recognitionofRNAviruses.TLR7,TLR8,and

TLR9areexpressedinendosomesandrequireendosomalmaturationforefficientsignaling.siRNArecognitionbyTLR7,TLR8,andTLR9resultsin

activationofNF-kBandIRFs,whichinduceinflammatorycytokinesandIFNs,respectively.TLR7andTLR8mediatetherecognitionofsiRNAsina

sequence-dependentmanner:preferentiallyU-andG-richsiRNAsarerecognized.RecognitionofsiRNAsbyTLRstakesplaceintheendosome,

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beforethesiRNAsenterthecytoplasm.Therefore,ifsiRNAscanenterthecytoplasmavoidingtheendosome,theyshouldbypasstheactivation

ofimmunesystemsbutstillmediategenesilencing.

Secondary screening: confirmation of candidates

ThegoalofthesecondaryscreenistoconfirmthatthephenotypeobservedisaspecificeffectofsiRNA-inducedtargetknockdownandnotan

off-targeteffectdependentontheprotocolorareagent.BecausethesiRNAdeliveryconditionswereoptimizedforapositivecontrolsiRNA,the

siRNAsthattargetcandidatehitsshouldbefurthertestedforsilencingefficiencyinsecondaryscreens[6],usingtechniquesdescribedinChapters

10and11.Iftherearealargenumberofcandidatehits,optimizationexperimentsmayhelpprioritizewhichgenesshouldbefollowedupfirst.

Confirming that phenotypes are due to reduced expression of the intended target

Test multiple siRNAs. Tominimizefalsepositiveandfalsenegativeresults,werecommendusingtwoormoresiRNAsthattargetdistinctregions

ofeachgeneanddeliveringeachsiRNAintriplicate,fortheinitialscreeningexperiment.AlthoughthenumberofsiRNAstargetingthesamegene

requiredtoconfirmacandidatehitcanbedeterminedstatisticallybyestimatingtheprobabilitythatanyrandomsiRNAfromthelibrarywillscore

positivelyintheassayunderthechosenconditions(i.e.,thefalsepositiverate),mostresearchersconsideransiRNAtargetaconfirmedhit,ifat

leasttwodifferentsiRNAsthattargetthecandidateproducethesamephenotype.Iftheinitialscreenwascarriedoutusingmultipleindividual

siRNAspertarget,youmaywanttoyourprioritizefollowuptofirstincludeonlygenesforwhichatleasttwodistinctsiRNAsgiveaphenotype

outsidethesetthresholdsoftheassay.NotethatcandidatehitswhereonlyoneofthesiRNAsgivesthedesiredphenotypeshouldnotbeignored.

Reproduce the phenotype. Forwell-characterizedtargetproteins,antibodiesorsmallmoleculeinhibitorsmayexistandcanbeusedtomimic

orcounteracttheeffectofthesiRNA[7].Toconfirmthatahitisassociatedwithaspecificpathway,additionalphenotypes(e.g.,up-ordown-

regulationofothergenes,increaseordecreaseinproteinmodification),thatareknowntooccurwhenthepathwayofinterestisperturbed,can

beassayedaftersiRNAtreatment[8,9].Theseadditionalassaysshouldbebasedonexistingknowledgeofthebiologicalprocess.Tofurtherstudy

complexcellularprocesses,someresearchershavebeguntodevelopautomated,microscopy-basedassaystosimultaneouslyanalyzefluorescent

markerslinkedtoantibodiesorligandstoassessrelativeintensityandcellularlocalizationovertime[6,10].

Perform a rescue experiment. FunctionalvalidationofRNAiexperiments is supportedbysuccessful siRNA-refractory rescueexperiments. In

theseexperimentsoverexpressionfromcDNAconstructsoftargetmRNAsthatarenotrecognizedbygene-specificsiRNAsareusedtorestorethe

wildtypephenotype.Forexample,acDNAwithasilentmutation(s)inthesiRNAtargetsequenceisexpectedtorescuethephenotypecaused

bysiRNA-inducedreductioninendogenousgeneexpression.Alternatively,aphenotypecausedbysiRNAsthatrecognizes5’or3’untranslated

sequencescanberescuedbyexpressingacDNAofthetargetgenethatcontainsonlycodingsequences[11].Recently,bacterialartificialchromo-

sometransgenesisinculturedcellshasbeenusedtocreateRNAi-resistanttransgenes[12].

Measure mRNA and protein levelsAsmentionedabove,confirmationofpositivehitsinvolvesverifyingthattheobservedphenotypeisspecificallyduetoreducingtheexpression

ofthetargetgene.Wheneverpossible,determiningsiRNA-inducedreductioninbothmRNAandproteinlevelsisrecommendedtohelpinterpret

yourdata.Inbothcases,targetmRNAorcorrespondingproteinlevelsshouldbeassessedrelativetothatofsamplestreatedwithnontargeting

negativecontrolsiRNAs.

IftargetmRNAlevelsarenotappreciablyreduced,yetthesiRNAinducesapositiveresultintheassay,itispossiblethattheobservedpheno-

typeiscausedbyanoff-targeteffect.However,inthisscenario,thecorrespondingproteinlevelsmaybereducedwithoutobservingareduction

intargetmRNAlevels.Therearetwopossiblereasonsforthisobservation:thesiRNAcouldbeactingasamicroRNAandinhibitingtranslation

ratherthantargetingitscognatemRNAfordestruction,ortheRNAassaymaynotaccuratelymeasuretargetmRNAlevels.AnRNAassaymayfail

toaccuratelyreflectmRNAknockdownifthetargetmRNAcleavageproductisrelativelystableoriftheassayisdesignedincorrectly(forexample

itdoesnotmeasureoneormoretargetedsplicevariantsorqRT-PCRnormalizationwasinaccurate).Ineithercase,itisagoodideatomeasurethe

reductionintargetproteinlevelsaftersiRNAdelivery.IfknockdownisnotobservedateitherthemRNAorproteinlevel,yetthesiRNAinducesa

distinctphenotype,itismorelikelythatthephenotypeistheresultofanoff-targeteffect.

siRNA screening6



Onenoteofcaution:werecommendthatcellviability(i.e.,cellnumber)bemonitoredinadditiontosiRNAinducedreductionintargetgene

expressionlevelsincasethetargetgeneisessentialforcellsurvival.LowcellviabilitymaycomplicatemRNAandproteinmeasurementsandlead

toerroneousresults.Onewaytostudyessentialgenesistousereporterassaysasdescribedbelow.Alternatively,itmightbepossibletomonitor

theeffectsofsiRNAtreatmentatshortertimepointsbeforesignificantcelldeathisobservedinyourcultures.

Assess mRNA levels. AssessingmRNAlevelsofthetargetgeneisadirectwaytomonitorsiRNA-inducedgenesilencing.QuantitativeRT-PCR,

Northernblots,branchedDNAassays,ormicroarrayanalysescanbeusedtomeasuremRNAlevels.qRT-PCRisthemostcommonmethod,inpart

becauseofitshighsensitivityandrapidity;however,replicatesandnormalizationcontrolsneedtobecarefullyassessedtoavoidmissingslight

changesinlevelsofgeneexpression.

Assess protein levels. Measuringprotein levelsoffersanotherdirectwayto link reducedgeneexpressiontothe loss-of-functionphenotype.

Rememberthattheoptimaltimeformeasuringproteinlevelsisbasedonthehalf-lifeoftheprotein.Also,themagnitudeofthedecreaseinpro-

teinexpressionrequiredtodetectaloss-of-functionphenotypewilldifferamongproteinsandmayvarywithcellcycle.Asdescribedinprotein

basedassaysonpage91,therearemanyassaysforquantitatingproteinlevels.Thechoiceofmethoddependsonthespecifictargetandavailability

ofappropriateequipment,assays(e.g.,protocolstomeasureenzymeactivityoriontransport)orantibodies.

Reporter gene assays. Wheneverpossible,detectionofendogenousproteinlevelsispreferredbecauseitprovidesadirectlinktochangesthat

occurinyourmodelsystem.However,reporterassays,whichmonitorexogenousproteins(expressionorenzymeactivity,canbeausefulalterna-

tiveforvalidatingcandidatehitswithrelativelylowexpressionorwithoutavailableantibodies.

Forexample,cotransfectionofsiRNAwithplasmidsexpressingthetargettranscriptmaybehelpfulforstudyinglowabudancemRNAtar-

gets[11].BecausetransfectionconditionsareoftennotthesameforplasmidsandsiRNA,celllinesstablytransfectedwiththeplasmidmaybe

requiredinsomecasestotestsiRNAeffects.

Reporterconstructs facilitate theuseoffluorescent (e.g.,GreenFluorescentProteinorRedFluorescentProtein) [12,3]orenzymatic (e.g.,

luciferase or β-galactosidase) assays to monitor changes in target gene expression. This approach can involve expression of a fusion protein

containingreporterandtargetsequences[7].Alternatviely,theconstructcanbedesignedtogenerateachimericmRNAinwhichatranslation

stopcodonseparatestheopenreadingframeofthereportergenefromthesiRNAtargetsequencesothatonlythereportergeneistranslated.

Insteadofreportergenes,anotherapproachinvolvesfusingepitopetages(e.g.,glutathione-S-transferase,Myc,hemagglutinin,6-histidine,

orFLAG®epitopetags)tothetargetproteintoenabledetectionwithwell-characterizedcommerciallyavailableantibodies[7,11,14].

Chapter references1. PuriN,WangX,VarmaRetal.(2008)Nuc Acids Symp SeriesNo.52.25–26.

2. EcheverriC,PerrimonN(2006)Nat Rev GenetApr11,epubaheadofprint.

3. (2003)Nature Cell Biology5:489–490.

4. (2005)Ambion Technotes™11.6:24–25.Alsoatwww.ambion.techlib/tn/116/19.html.

5. BrownD,ByromM,KrebsJetal.(2006)Ambion Technotes™12.1:23–25.Alsoatwww.ambion.techlib/tn/121/11.html.

6. SachseC,KrauszE,KronkeAetal.(2005)Methods Enzymol392:242–277.

7. BrummelkampTR,NijmanSM,DiracAMetal.(2003)Nature424(6950):797–801.

8. Aza-BlancP,CooperCL,WagnerKetal.(2003)Mol Cell12(3):627–637.

9. SilvaJM,MizunoH,BradyAetal.(2004)Proc Natl Acad Sci U S A101(17):6548–6552.

10. SachseC,EcheverriCJ(2004)Oncogene23(51):8384–8391.

11. HsiehAC,BoR,ManolaJetal.(2004)Nucleic Acids Res32(3):2333–2340.

12. KittlerR,PelletierL,MaCetal.(2005)Proc Natl Acad Sci U S A102(7):2396–2401.

13. KumarR,ConklinDS,MittalV(2003)Genome Res13(10):2333–2340.

14. SandyP,VenturaA,JacksT(2005)Biotechniques35(2):215–224.

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siRNA screening6

Section II—Products for RNA Interference

Chapter 7—siRNA technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40

Chapter 8—Vector-based RNAi technologies . . . . . . . . . . . . . . . . . . . . .53

Chapter 9—RNAi delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Chapter 10—RNA interference controls . . . . . . . . . . . . . . . . . . . . . . . . 79

Chapter 11—Measuring knockdown . . . . . . . . . . . . . . . . . . . . . . . . . .88

Chapter 12—RNAi services. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

Chapter 13—Overview of miRNAs. . . . . . . . . . . . . . . . . . . . . . . . . . . .98

Chapter 14—Optimized miRNA profiling. . . . . . . . . . . . . . . . . . . . . . . 115


Section II Products for RNA Interference

CHAPTER 7

siRNA technologiesIntroduction to nonvector methods for transient RNAiFortransientknockdownexperiments,synthetic,nonvectorapproachesoffersignificantadvantagesovervector-basedmethodsforRNAideliv-

ery. Inparticular,nonvectorexperimentsaretypicallyeasier todesignandperformandcanresult inhigher levelsof transientknockdown. In

addition, recent improvements inRNAidesignhave increasedthe likelihoodofachievinghigh-levelknockdownaftertestingonlya fewRNAi

molecules.Consequently,usingsyntheticallygeneratedRNAduplexesisthemostpopularmethodforconductingRNAiexperiments.

Concerns about nonspecific effects. AlthoughtheuseofsyntheticRNAimoleculesisaneasy,effectivemethodofinhibitinggeneexpression,

there issomeconcernabout thespecificityof thesemolecules.The introductionofsomesiRNAduplexes intomammaliancellscanresult in

phenotypicchangesunrelatedtoinhibitionofthetargetgene,therebygeneratingfalse-positiveresultsthatarenonspecific.Nonspecificeffects,

whicharedue to regulationofunintendedgene targets,aremostcommonly referred toasoff-targeteffects,andmaybedue tosequence-

specificmotifsthatgenerateaptamer-likeeffects,suchasactivationofstressresponsepathways.siRNAduplexeswithpartialhomologytoother

targetsmayalsocontributetooff-targetactivity.Geneprofilingexperimentshaveshownthatduplexeswithpartialhomologytoothertranscripts

cancleavethetargetoractlikemicroRNA(miRNA)andinhibittranslation[1–3].SpecificitystudieshaverevealedthatsiRNAduplexescanhave

varyingactivitiesdependingonthenumber,position,andbase-paircompositionofmismatcheswithrespecttothetargetRNA[4].Whilethese

studieshaveshownthatduplexeswithpartialhomologytoatargetcanregulategeneexpression,therulesfordesigningduplexestoeliminate

off-targeteffectsarenotcompletelyunderstood.

Unwanted interferon responses. It is generally accepted that siRNA duplexes with fewer than 30 base pairs evade recognition by the pro-

teinsmediatingthemammalianantiviralresponse.However,mountingevidencesuggeststhatsomesiRNAscanactivateinterferonandstress

responsepathways.Inmammaliancells,double-strandedRNAs(dsRNAs)arerecognizedbydsRNA-bindingproteinsandToll-likereceptors,lead-

ingtoglobalshutdownofproteinsynthesisandactivationoftheinterferonresponse.Recently,somesiRNAscontainingsequence-specificmotifs

thatinducetheinterferonresponsehavebeenidentified[5,6].EliminatingthesesequencemotifsduringthesiRNAdesignprocessisonestrategy

toavoidinducingstressresponsepathways.Nonetheless,othersequencemotifsmightexist,andmethodstoscreenforinducingthesemotifs

arelargelyuncharacterized.

Enhancing specificity. Advanced sequence design algorithms can be applied to reduce nonspecific effects due to off-target regulation or

sequence-specificactivationofstressresponsepathways.Thissolutionrequiresadetailedunderstandingoftherulesfordesigninghighlyprecise

duplexes.Analternativeapproachtoenhancingspecificityliesintheuseofchemicalmodifications.

Ambion® Silencer® Select siRNA and Stealth RNAi™ siRNA

RNAinterference,thebiologicalmechanismbywhichdouble-strandedRNA(dsRNA)inducesgenesilencingbytargetingcomplementarymRNA

fordegradation,isrevolutionizingthewayresearchersstudygenefunction.Forthefirsttime,scientistscanquicklyandeasilyreducetheexpres-

sionofaparticulargeneinmammaliancellsystems,oftenby90%orgreater,toanalyzetheeffectofthatgeneoncellularfunction.

Proven siRNAs for in vitro and in vivo analyses. InvitrogennowoffersAmbion®products.Asaresult,youwillfindthetwobestsiRNAtechnologies

availableinoneplace.Silencer®SelectsiRNAsarethebest-performingsiRNAsforin vitro studies,andareavailableinavarietyofformatsincluding

preplatedcollectionsandcustomlibrariestosimplifyscreeningexperiments.StealthRNAi™siRNAsarethegoldstandardforin vivo applications,

andhavebeenproveneffectiveinhelpingtomeetthedemandsof in vivo experiments.Together,thesetoolsallowyourexperimentstoyield

accuratedataandconclusions—fromhypothesistodemonstratedphenotype.

Silencer® Select siRNAsThebestsiRNAsforin vitro applications

→ Classical21-mersiRNAswithenhancedchemicalmodificationsandimprovedalgorithm

→ Specificknockdown—novelchemicalmodificationsreduceoff-targeteffectsbyupto90%

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→ Effectiveknockdown—designedwithanall-newalgorithmthattypicallyimprovespredictionaccuracybymorethan25%

→ Reliableresults—demonstratedimprovementsinconsistencyandreliabilityofphenotypicresults

→ Unparalleledpotency—upto100-foldmorepotentthancurrentlyavailablesiRNAs

→ Guaranteedconfidence—100%guaranteedtosilence,thebestguaranteeintheindustry

Cleaner, more consistent data. The Silencer®Select siRNAs incorporate the latest improvements in siRNAdesign,off-targeteffectprediction

algorithms,andchemistry.Asaresult,theyallowunrivaledsilencingconsistency,potency,andspecificity,andfewerfailedexperiments,enabling

cleaner,moreconsistentphenotypicdata.

Design algorithm improves siRNA effectiveness. CurrentsiRNAdesignalgorithmspredictsiRNAsthat induce70%targetmRNAknockdown

withonly~80%confidence.ManyRNAiapplicationsdemandbetterefficiency.TheSilencer®SelectsiRNAdesignalgorithmwasdevelopedusinga

powerfulmachinelearningmethod.PerformancedatafromthousandsofsiRNAswereanalyzedtobetterunderstandthelinkbetweenansiRNA’s

sequence,targetlocation,andthermodynamicpropertiesanditssilencingefficiency(Figure7.1).TheresultismoreeffectivesiRNAs(Figure7.2).

The Silencer® Select siRNA design algorithm. Thedesignalgorithmincorporatesmorethan90differentsequenceandthermodynamicparam-

eterstoincreasepredictiveaccuracy28%overprevious-generationsiRNAdesignalgorithms.TheresultissiRNAsthatareupto100-foldmore

potentthanothersiRNAs(modifiedandunmodified),allowingahigherpercentageof“on-target”phenotypes.

Phenotype from

siRNA 1

Phenotypefrom

siRNA2

Phenotypefrom

siRNA3

Chemical modifications

Bioinformatic filters

Design algorithm

Silencer® Select siRNAs Increased confidence

in results

More consistent knockdown Better potency

Reduced toxicity Better specificity

Figure 7.1 The concept behind Silencer® Select siRNAs.

Figure 7.2. Silencer® Select siRNA design algorithm significantly improves effective siRNA prediction accuracy. TheSilencer®SelectsiRNAdesignalgorithmwasusedtodesign155siRNAsto40differenttargets.ThesesiRNAsweretestedsidebysidewithsiRNAsdesignedusingthepreviousalgorithmat5nMinHeLacells.mRNAknockdownwasmeasured48hrposttransfectionviaqRT-PCRusingTaqMan®GeneExpressionAssays.ResultsareexpressedaspercentofmRNAremainingcom-paredtoSilencer®NegativeControl#1siRNA–treatedcells.TheinsetshowsthepercentageofsiRNAsthatelicited≥70%and≥80%mRNAknockdown.

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101 106 111 116 121 126 131 136 141 146 151

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More potent knockdown, fewer off-target effects. HighersiRNAconcentrationsareknowntoincreaseoff-targeteffects[1,7].Theneedformore

potentsiRNAsthatcanbeusedatlowerconcentrationswasakeyconsiderationinthesiRNAdesignalgorithmimprovementprocess.TheSilencer®

SelectsiRNAsshowincreasedpotencyandfeweroff-targeteffectscomparedtofirst-andsecond-generationsiRNAdesigns:

→ Upto100xmorepotentthancompetitorsiRNAs(Figure7.3)

→ Canberoutinelytransfectedat≤5nMandretaintheirsilencingpower

→ Feweroff-targeteffectswhenusedattheselowerconcentrations

→ ReducedcostperexperimentthansiRNAsusedathigherconcentrations

More consistent yield of silenced phenotypes. ThemaingoalofanRNAiexperimentistoexaminethebiologicaleffectofknockingdowna

targetofinterest,oftenwithacell-basedassay.However,toelicitthatphenotype,someminimumthresholdlevelofknockdownisrequired,and

thisthresholdlevelwillvarydependingonthetarget.Silencer®SelectsiRNAs:

→ Morereliablyelicitmaximumknockdownlevels(Figure7.4)

→ Moreconsistentlyreachthethresholdlevelofknockdownrequiredtoseealoss-of-functionphenotype

→ Inside-by-sidetests,resultinahigherpercentageofexpected,silencedphenotypesthansiRNAsfromothervendors

Figure 7.3. Silencer® Select siRNAs provide up to 100x higher potency compared to other siRNAs. Silencer®SelectsiRNAsto10different targetsandsiRNAs fromtwoothersuppliers tothesame10differenttargetswereindividuallytransfectedintoHeLacellsintriplicateattheindicatedsiRNAconcentration.mRNAknockdownlevelsweretested48hrlaterasdescribedinFigure7.4.AveragepercentmRNAremaining isshownforeachsetofsiRNAs.

Figure 7.4. Silencer® Select siRNAs elicit expected phenotype at a higher rate than other siRNAs. siRNAs to7gene targetswithwell-understoodRNAi-inducedphenotypeswere indi-viduallytransfectedat3nMandphenotypesmeasured48hrlater.Eachbarrepresentsthepercentof siRNAs thatgave theexpected, silencedphenotype. siRNAs toBUB1B,AURKB,WEE1,andPLK1wereassessedusingamultiparametriccellgrowth/apoptosisassay in U2OS human osteosarcoma cells. siRNAs to HMGCR, LDLR, and FDFT 1 wereassessedusinganLDLuptakeassayinHUH7humanhepatomacells.

SILENCER® SELECT siRNA KNOCKDOWN GUARANTEE

→ Buy2Silencer®SelectPre-designedsiRNAstoatarget;bothareguaranteedtoknockdowntargetgeneexpressionby≥70%

→ Buy3Silencer®SelectPre-designedsiRNAstoatarget;2of3areguaranteedtoknockdownby≥80%

→ Silencer®SelectValidatedsiRNAsareguaranteedtoknockdownby≥80%

Fordetails,visitwww.invitrogen.com/rnai.

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Bioinformatic filtering improves siRNA specificity. AlthoughusingsiRNAsatlowconcentrationsdecreasesoff-targeteffects,furtherspecificity

gainscanbemadeusingbioinformaticfilteringtopredictandeliminatepotentially“bad”siRNAs.TheSilencer®SelectsiRNAdesignincludesa

5-stepbioinformaticfilteringprocess(Figure7.5),which:

→ RemovessiRNAswithahighpropensityforoff-targeteffects

→ UsestheSilencer®SelectsiRNAToxicityClassifier,whicheliminatessequencespredictedtoelicitanoff-targetapoptoticphenotype

→ MinimizesmiRNApathway–relatedoff-targeteffectsbyremovingsiRNAswithseedregionsthatresemblenaturallyoccurringmiRNAsand

selectingsiRNAswiththefewestseedregionmatchesinthe3’UTRsofoff-targettranscripts

Chemical modifications enhance guide strand bias. Strongguidestrandbias,wheretheguidestrandofthesiRNAispreferentiallytakenup

intotheRISCoverthepassengerstrand,isimportantbothformaximizingsiRNAsilencingpotencyandfordecreasingpassengerstrand–related

off-targeteffects(Figure7.6).AlthoughincorporatingtherightsiRNAdesignparameterscanhelp,siRNAdesignaloneisnotsufficienttoensure

strongguidestrandbias.TheSilencer®SelectsiRNAs:

→ Consistentlyenhanceguidestrandbias,whichcorrelatesstronglytoknockdownefficiency

→ Preventthepassengerstrandfrominducingsilencing,whichservestoreduceoff-targeteffects

→ ResultinnolossinsiRNAsilencingpotency;inmanycasesanimprovementisseen

Figure 7.5. Five-step bioinformatic filtering process that eliminates siRNAs predicted to elicit off-target effects.

% Luciferase activity remaining

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Mismatch Filter

Silencer® Select siRNA Toxicity Classifier

Natural miRNA Seed Region Filter

Antiviral Response Motif Filter

siRNA Seed Region Ranking

Advanced Silencer® Select siRNA

Silencer® Select siRNA Design Algorithm

Figure 7.6. Silencer® Select siRNAs show enhanced guide strand bias.Luciferase reportergeneconstructswithsiRNAtargetsclonedineitherthesense(guidestrandtarget)orantisense(passenger strand target) orientation were cotransfected with the corresponding siRNAand a β-galactosidase–encoding control vector. Luciferase and β-galactosidase assayswereperformed72hourslater,andknockdownforeachstrandwascalculatedrelativetonegativecontrolsiRNA–transfectedcells.Theaverageratioofguidetopassengerstrandknockdownisplottedagainstluciferaseknockdownfor6Silencer®Selectand36competi-torsiRNAs.

7siRNA technologies



More consistent, reliable data. Sequence-specificoff-targeteffectsareoneoftheprimaryreasonsforfalsepositiveresultsinRNAiexperiments.

Inadditiontothepotencyimprovementsaffordedbythenewalgorithmandstate-of-the-artbioinformaticfilteringcriteria,Silencer®Select siR-

NAsincorporatenovelmodificationsthatimprovesiRNAspecificity,allowingcleaner,moreconsistentcellbiologydata.Thesemodifications:

→ Reduce the number of nontargeted, differentially expressed genes detected by gene expression array by up to 90% as compared to

unmodifiedsiRNAs(Figure7.7)

→ Resultinadramaticreductionofoff-targetphenotypesasmeasuredbymultiparametriccell-basedassays(Figure7.8)

→ Donotnegativelyimpactsilencingefficiencyandthereforedonotcompromisetheexpectedon-targetphenotypes

Figure 7.7. Silencer® Select siRNA modifications reduce the number of off-target, diff erentially expressed genes. ThreenegativecontrolsiRNAswithandwithouttheSilencer®Selectmodifications were individually transfected in quadruplicate into HeLa cells at30 nM. RNA was extracted and analyzed on an Affymetrix Human Genome U133Plus2.0Arrayintriplicate.They-axis indicatestheaveragenumberofdifferentiallyexpressedgenes—thoseshowing≥2-foldchangeinexpressioncomparedtomock-transfectedsamples.

30

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Apoptosis Phenotypes

Off-target phenotypes elimated by modi�cations

WEE1

PLK1

*in vivoreferstoresearchuseinsmallanimals.Thisproductisnotintendedforuseinhumans.

Figure 7.8. Silencer® Select siRNA modifications reduce off-target effects and yield more reli-able phenotypic data. 53different siRNAs, includingolderdesignspreviouslynotedtoelicitoff-targetphenotypes,were transfected intoU2OScellsat30nM inbothunmodified and Silencer® Select modified formats. Mitosis and apoptosis weremeasured 48 hours later. Data are expressed relative to negative control siRNA–transfectedcells.Black=similarmitosis/apoptosislevelsascontrol.Green=down-regulation. Red = up-regulation. Note that the expected mitotosis and apoptosisphenotypesforPLKandWEE1siRNAsarepreservedwiththemodifications.Incon-trast, the off-target apoptotic phenotypes elicited by 10 unmodified siRNAs werecompletelyeliminatedwithadditionoftheSilencer®Selectmodifications.

Stealth RNAi™ siRNAThegoldstandardforin vivo*RNAiapplications

→ Award-winningdesignalgorithm—allowsthehighestratesofknockdownsuccess

→ Highstabilityagainstnucleases—duplexesreachtheirtargetintactandreadytoinitiateknockdown

→ Noinductionoftheinterferonresponse—correlatephenotypeswithknockdownactivityinsteadoftoxicity

→ EasytrackingofadministeredRNAiduplexes—measurebiodistributioneffectively

→ Knockdowneffectivenessandstability—noneedtocompromiseonefortheother

In vivo RNAiexperimentsaremorechallengingthantheir in vitro counterpartsdueto thedemandsof thecellularenvironment.Theseadded

challengesnecessitatetheuseofthehighest-qualitymaterialstoobtainmeaningfulresults,andadeliveryreagenttotransfectcellsintheorgans

targetedinthesubjectanimal.

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StealthRNAi™siRNAmoleculesarechemicallymodified,blunt-ended,25-merdouble-strandedduplexesthatarerecognizedbytheRNA-

inducedsilencingcomplex(RISC)tomediateinhibitionofatargetgene.ProprietarychemicalmodificationsallowStealthRNAi™siRNAtoover-

comemanyin vivo–specificobstacles,allowingeffectivenessandstabilityinin vivo applications.

Specific and effective knockdown. InRNAiexperiments,itisnecessarytominimizeoff-targeteffectssothatobservedphenotypescanbecor-

rectlyattributedtotargetknockdown.WithstandardsiRNAduplexes,bothstrandsareabletoparticipateintheknockdownreaction,increasing

thechancesofoff-targeteffects.WithStealthRNAi™siRNAduplexes, thesensestrand ischemicallymodifiedtoprevent it fromcontributing

toRNAiactivity.StealthRNAi™siRNAduplexesaredesignedwithInvitrogen’saward-winningBLOCK-iT™RNAiDesigner(www.invitrogen.com/

rnaidesigner),whichusesSmith-WatermanalignmenttocomparetheRNAduplexregiontotheentiregenome,includinguntranslatedregions.

Thisdesignstepminimizesthepotentialforoff-targeteffectsandallowsaveryhighpercentageofexperimentallysuccessfulduplexes(Figures

7.9and7.10).

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Figure 7.9. Stealth RNAi™ siRNA exhibits increased target specificity. Sequences1and2weredesignedaseithersiRNAorStealthRNAi™siRNAduplexesagainstatargetgene.OnlytheantisensestrandofaStealthRNAi™siRNAduplexcanentertheRISCforspecifictargetingofmRNA,whereaseitherstrandofsiRNAcanentertheRISC,increasingthepossibilityofnonspecificsilencing.

Figure 7.10. Success rate of Stealth RNAi™ siRNA design exceeds 90%. InvitrogenhasbeengeneratingdatatoevaluatetheefficacyofalargenumberofStealthRNAi™siRNAduplexessinceinitiationofitsextensiveValidatedStealthRNAi™siRNAprogramin2004.Thisdatasetwasderivedfrom1,000StealthRNAi™siRNAduplexestargetedto171genesgeneratedusingtheBLOCK-iT™RNAiDesignerfrom2004.KnockdownwasmeasuredusingtheRNAitargetscreen-ingsystem,whichassessesknockdownattheproteinlevel.Over90%oftheseduplexes(boundedbygreenbox)resultedin≥70%knockdown,and30%(boundedbyyellowbox)resultedin≥97%knockdown.Theseresultswereobtainedusing2nMStealthRNAi™siRNAduplexes.Typically,10to100nMisusedforthesetypesofexperiments;thehigherendofthisrangecanleadtooff-targeteffects.Thedatawereanalyzedateachpositiontodeterminethedifferencesbetween“good”and“bad”StealthRNAi™siRNAsequences.ThisinformationwasthenusedtotrainthealgorithmandhasbeenincorporatedintothelatestversionoftheBLOCK-iT™RNAiDesigner(www.invitrogen.com/rnaidesigner).

7siRNA technologies



Stable in the presence of nucleases. NucleasescapableofdegradingsiRNAduplexescontributetothechallengingin vivo experimentalenvi-

ronment.NucleaseresistancehelpsensurethatsiRNAduplexesreachtheirintendedlocationintactandthattheyarecapableofinitiatingthe

knockdownreaction.StealthRNAi™siRNAduplexeshaveproprietarychemicalmodificationsthatconferremarkablestabilityfor in vivo studies

(Figure7.11);standardsiRNAslacksuchmodifications,makingthemmoresusceptibletodegradationbynucleases.

No induction of the interferon response. Theinterferonresponseisapowerfulimmunereactionthatcanmakeanalysisofknockdownnearly

impossibleduetononspecifictoxiceffects.Inductionoftheinterferonresponsecanleadtoseverelyalteredglobalgeneactivityandmakephe-

notypicassessmentdifficult.StandardsiRNAhasthepotentialtoactivatethisinnateresponse,settingoffdefensesystemsusuallyusedtocombat

viruses. Chemical modifications in Stealth RNAi™ siRNA molecules abolish the immunostimulatory response observed with some sequences.

Stealth RNAi™ siRNA complexes do not induce the interferon response (Figure 7.12), providing confidence that observed phenotypes can be

attributedtospecifictargetknockdownandnottononspecifictoxicity.

Tracking delivered duplexes. TrackingthebiodistributionofadministeredRNAiduplexesisnecessarytoensurethatthedeliveredmaterialhas

targetedthedesiredtissues. Invitrogenusesthe industry-leadingAlexaFluor®fluorescentdyesto labelStealthRNAi™siRNAduplexes for this

purpose.Thesedyesareidealforin vivo RNAiapplications,duetotheirsuperiorbrightnessandphotostability.Duplexescanbetrackedinthree

differentways:aStealthRNAi™siRNAduplexcanbedirectlylabeledwithanAlexaFluor®dye,itcanbebiotinylatedanddetectedwithlabeled

streptavidin,oralabeledcontrolduplexcanbemixedwithatarget-specificStealthRNAi™siRNAduplex(Figure7.13).DirectlabelingofStealth

RNAi™siRNAduplexesdoesnotaffecttheirpotency.

Together,theSilencer®SelectsiRNAandtheStealthRNAi™siRNAtechnologiesprovideasuperior,complete,androbustsolutionforyour

RNAistudiesbothin vitro andin vivo.FormoreinformationontheseandotherproductsforRNAistudies,visitwww.invitrogen.com/rnai.

21-mer siRNA Stealth RNAi™ siRNA 21-mer siRNA Stealth RNAi™ siRNA

Mouse serum Human serum

0 4 8 24 48 720 4 8 24 48 720 4 8 24 48 72 0 4 8 24 48 72

Figure 7.11. Chemical modification makes Stealth RNAi™ siRNA more stable for in vivo applications. Stealth RNAi™ siRNA duplexes are chemically modified toenhancestabilityagainstnucleasesinserum.Unmodified21-merdsRNAsequencesandcorrespondingStealthRNAi™siRNAsequenceswereanalyzedat0,4,8,24,48,and72hrfollowingincubationineither10%mouseorhumanserum.SampleswereseparatedonaNovex®15%TBE-ureapolyacrylamideprecastgelandstainedwithmethyleneblue.ThesedatademonstratethatStealthRNAi™siRNAduplexesremainintact,whereasunmodified21-merdsRNAsequencesweresusceptibletopronounceddegradation.

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Figure 7.12. Stealth RNAi™ siRNA avoids the interferon stress response so that data can be confidently correlated with knockdown activity. TailveinsofBalb/cmice(threemicepertreatment)wereinjectedat5mg/kgand10mg/kgofStealthRNAi™siRNAduplexes.After6hr,serumwascollectedandprocessedtomeasureinterferonmarkersusingtheInvitrogenCytokineMouse20-PlexPanelfortheLuminex®instrument(Cat.No.LMC0006).

→ No treatment

3 mice per treatment:

6 hr

Luminex®

assays

→ CpG in PBS (pos. ctrl)→ 37 bp dsRNA→ Stealth RNAi™ siRNA in PBS

IL-1αIL-1βIL-2IL-4IL-5IL-6IL-10IL-12p40IL-12p70IL-13IL-17TNF-αIFN-γ

G-CSFGM-CSFFGF-basicVEGFPDGF-BBMIP-1αMIP-1βMIP-3βKCIP-10MCP-1MIGRANTES

Cytokine 10-plexTh1/Th2 6-plexIn�ammatory 4-plexChemokine 5-plexGrowth factor 4-plexCytokine 20-plex

19 markers Multiplex panels

Sacri�ce and collect serum

Rel

ativ

e va

lues

(log

sca

le)

0.1

1

10

100

37 bpdsRNA

2.5 mg/kg

Stealth RNAi™

siRNA2.5 mg/kg

CpGNotreatment

IL-6

IP10

MIP-1α

IL-12

MCO-1

TNF-α

ProtocolR

af-1

/GA

PD

H r

atio

Raf-1 +BLOCK-iT™

Raf-1 biotinRaf-1 Alexa Fluor® 647

Raf-1Control

0.2

0

0.4

0.6

0.8

1.0

1.2

1.4

Raf-1RNAi–Alexa Fluor® 647

Raf-1 RNAi, biotinylated Alexa Fluor® 488–streptavidin

Raf-1 RNAi + BLOCK-iT™ Alexa Fluor® 555

Figure 7.13. Labeled Stealth RNAi™ siRNA exhibits potent knockdown. MDA-MB-435cellsweretransfectedwith25nmolunlabeledStealthRNAi™siRNAduplexes,orStealthRNAi™duplexeslabeledwithanAlexaFluor®dyeorbiotin.IneachcasetheduplexestargetedRaf-1.Knockdownwasassessedat>90%byreal-timePCR;deliveryisdemonstratedthroughfluorescenceimaging.Regardlessofthelabelingstrategy,StealthRNAi™siRNAknockdownresultswereconsistent.

7siRNA technologies



siRNA selection guidesiRNA type Description Technology Recommended use

Pre-designed/Validated(tubes)

StealthSelectRNAi™siRNAandValidatedStealthRNAi™siRNA

• Modifiedforserumstabilityandimprovedspecificity

• Pre-designedforhuman,mouse,andratgenes• Validatedforsomehumangenes

Modified25-mer

• Suitableforallapplications• Particularlygoodforin vivoapplications

Silencer®SelectPre-designedandValidatedsiRNA

• Improvedpotencyandguaranteedknockdown• Modifiedforimprovedspecificity• Pre-designedforhuman,mouseandratgenes• Validatedforsomehumangenes

LNA®modified21-mer

• Suitableforallapplications• ParticularlygoodforcellcultureandRNAiscreen-

ingwork

Silencer®Pre-designedandValidatedsiRNA

• Pre-designedforhuman,mouse,andratgenes• Validatedforsomehumangenes

Unmodified21-mer

• Lower-costalternative,butwithoutsomeoftheperformancebenefitsofthenewertechnologies

• Suitableforallapplications

Pre-madesets/collections

Made-to-StockSilencer®SelectsiRNALibraries

• Pre-definedsetsofSilencer®SelectsiRNAsforthehumangenomeandfunctionalgeneclasses

• 0.25nmolofeachsiRNA• Readyforimmediatedelivery

LNA®modified21-mer

• Forscreeningofpre-definedSilencer®SelectsiRNAsetssuchashumankinases,GPCRs,drug-gableandwholegenome

Made-to-StockSilencer®siRNALibraries

• Pre-definedsetsofSilencer®siRNAsforthehumanandmousegenomesandfunctionalgeneclasses

• 0.25nmolofeachsiRNA• Readyforimmediatedelivery

Unmodified21-mer

• Lower-costalternativetoSilencer®SelectsiRNALibraries,butwithoutsomeoftheperformanceenhancements

Plated

Made-to-OrderSilencer®SelectsiRNALibraries

• CustomsetsofSilencer®SelectsiRNAsat0.1to5nmol

• Pricedbyquoteandorderedoffline

LNA®modified21-mer

• Forscreening36to>10,000Silencer®SelectsiRNAs

• Submitagenelistorchooseoneofourfunc-tionalsets

• Multiplesizesandplatingoptions

Made-to-OrderSilencer®siRNALibraries

• CustomsetsofSilencer®siRNAsat1to5nmol• Pricedbyquoteandorderedoffline

Unmodified21-mer

• Lower-costalternativetoSilencer®SelectsiRNALibraries,butwithoutsomeoftheperformancebenefits

PlateSelect™RNAiw/StealthSelectRNAi™siRNA

• StealthSelectRNAi™siRNAinplatedformatat1nmolsize

• ConfigureplateswithPre-designedsiRNAorsiRNAsdesignedonlinewiththeBLOCK-iT™RNAiDesigner

Modified25-mer

• ForStealthSelectRNAi™siRNAexperimentsinplateswith1to>96siRNAs

• SpecifyasetsiRNAsusingonlineinterface• Madetoorder

PlateSelect™RNAiw/BLOCK-iT™siRNA

• BLOCK-iT™siRNAinplatedformatat1nmolsize• ConfigureplateswithsiRNAsdesignedonline

withtheBLOCK-iT™RNAiDesigner

Unmodified21-mer

• ForunmodifiedsiRNAexperimentsinplateswith1to>96siRNAsat1nmol

• SpecifyasetofsiRNAsusingonlineinterface• Madetoorder

Design-your-ownsiRNAs

StealthRNAi™siRNA• ChemicallymodifiedsiRNAdesignedusingthe

BLOCK-iT™RNAiDesignerModified25-mer

• ForgenesthatfalloutsideofourPre-designedsiRNAcollection

• Suitableforallapplications• Particularlygoodforin vivoapplications

Block-iT™siRNA• Traditional,unmodifiedsiRNAdesignedusingthe

BLOCK-iT™RNAiDesignerUnmodified21-mer

• ForgenesthatfalloutsideofourPre-designedsiRNAcollection

• Lowercostalternative,butwithoutsomeoftheperformancebenefitsofStealthRNAi™siRNAs

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siRNA type Description Technology Recommended use

Custom(youprovidethesequence)

StealthRNAi™siRNA• Modifiedforserumstabilityandimprovedspeci-

ficityModified25-merModified25-mer

• WhenyouhaveansiRNAsequenceandneeditmanufactured

Block-iT™siRNA• TraditionalsiRNA• Availablewithaselectionoffluorescentlabels

andmodifications

Choiceof21–25-mers

• WhenyouhaveansiRNAsequenceandneeditmanufactured—withorwithoutalabel

• Lower-costalternative,butwithoutsomeoftheperformancebenefitsofStealthRNAi™siRNAs

Controls

StealthRNAi™siRNAPositive&NegativeControls

• siRNAcontrolsforusewithStealthRNAi™siRNA• Fluorescentlabelsavailablewithsome

Modified25-mer

• AnytimeyouareusingaStealthRNAi™siRNA

Silencer®SelectPositive&NegativeControlsiRNAs

• siRNAcontrolsforusewithSilencer®SelectsiRNAsLNA®modi-fied21-mer

• AnytimeyouareusingaSilencer®SelectsiRNA

Silencer®Positive&NegativeControlsiRNAs

• siRNAcontrolsforusewithSilencer®siRNAs• Fluorescentlylabeledcontrolsavailable

Unmodified21-mer

• AnytimeyouareusingaSilencer®siRNA

CustomScrambledNegativeControlsiRNA

• siRNAcontrolsforBLOCK-iT™siRNAs• MatchyourscramblednegativecontroltotheGC

contentofyourunmodified21-mersiRNA

Unmodified21-mer

• AnytimeyouareusingaBLOCK-iT™siRNA

Silencer® Select Pre-designed siRNAsSilencer® Select Pre-designed siRNAs come with a 100% guarantee to silence their intended target, and are available for >98% of genes in the

human,mouse,andratgenomesinapurified,ready-to-useformat.siRNAsequenceinformationisalwaysprovided.

Silencer® Select Validated siRNAsSilencer®SelectValidatedsiRNAsareindividualsiRNAduplexesthathavebeenverifiedexperimentallytoreducetheexpressionoftheirindividual

targetgenes.EachsiRNAwasdesignedusingthesameeffectivealgorithmusedtodesignSilencer®SelectPre-designedsiRNAs.However,each

Silencer®SelectValidatedsiRNAhasalsobeenfunctionallyconfirmedandisguaranteedtoreducetargetgeneexpressionbyatleast80%when

measured48hoursposttransfection.

Custom Designed Silencer® Select siRNAsThe Silencer® Select siRNA design algorithm yields an extremely high percentage of effective siRNA sequences. Silencer® Select Pre-designed

siRNAsareimmediatelyavailableforallhuman,mouse,andratgeneslistedinthepublicRefSeqdatabasemaintainedbyNCBI.However,ifyour

needsextendbeyondourcurrentselectionofPre-designedsiRNAs,weofferSilencer®SelectCustomDesignedsiRNAs,whicharedesignedusing

thesamealgorithmandwiththesamemodificationsasourotherSilencer®SelectPre-designedsiRNAs.

OrderSilencer®SelectsiRNAsatwww.invitrogen.com/RNAi.

7siRNA technologies



Silencer® Select siRNA libraries

Pre-defined siRNA libraries plated to orderPre-defined,made-to-orderSilencer®SelectsiRNALibrariesareavailableforhuman:

→ Genome

→ Druggablegenome

→ Extendeddruggablegenome

→ Kinases

→ Phosphatases

→ GPCRs(non-olfactory)

→ Ionchannels

→ Nuclearhormonereceptors

→ Proteases

Human Genome siRNA Library (Consists of Part Numbers: 4397922 + 4397924 + 4397923)

Increasingly,researchersareperforminggenome-scalesurveysofgenefunction.TheSilencer®SelectHumanGenomesiRNALibraryV4isidealfor

thispurpose.Threeunique,non-overlappingsiRNAsareprovidedforeachof21,585humantargets.ThesiRNAstargetingthe“druggable”portion

ofthislibraryarearrangedbygenefunctionalclasstoenableeasyscreeningofimportantgenesubsetssuchaskinase,phosphatase,GPCR,etc.

ThissiRNAlibraryissuppliedin384-wellplates.

Human Druggable Genome and Human Extended Druggable Genome siRNA LibrariesWithsiRNAstargetingthemosttherapeuticallyrelevanthumangenesandconvenientlygroupedbytargetgeneclass,theSilencer®SelectHuman

DruggableGenomesiRNALibraryV4andSilencer®SelectHumanExtendedDruggableGenomesiRNALibraryV4arepopularchoicesformany

researchers.Thedruggablegenomesettargets9,032genes,whereastheextendeddruggablegenomecollectiontargetsthesame9,032genes

plusthe“ExtensionSet”foratotalof10,415genes,includingtranscriptionfactors.Pleasecontactusforthecompletelistoftargets.Theselibraries

areavailableinboth96-and384-wellplateformats.

Human Kinase siRNA LibraryBecauseoftheirimportanceincellsignalingandmanybiologicalpathways,kinasesarekeydrugtargetsandsubjectsofintensescrutiny.The

Silencer®SelectHumanKinasesiRNALibraryV4targets710humankinaseswiththreeindividualSilencer®SelectsiRNAspergene.ThissiRNAlibrary

enablessystematicyetcost-effectiveRNAistudiesofthesekeycellregulators.ValidatedsiRNAsareincludedwhereavailable.TheSilencer®Select

HumanKinasesiRNALibraryV4isprovidedin96-wellplates.

Human Phosphatase siRNA LibraryAlong with kinases, phosphatases are important regulators of biological pathways and cell signaling cascades. The Silencer® Select Human

PhosphatasesiRNALibraryV4targets298humanphosphataseswiththreeindividualsiRNAspergenesuppliedin96-wellplates.Thislibraryisthe

idealcompaniontotheSilencer®SelectKinasesiRNALibraryV4,enablingmoredetailedstudiesofbiologicalpathways.

Human GPCR siRNA LibraryG-protein–coupled receptors,orGPCRs,arekeycomponentsofmanysignal transductionpathwaysandalso representan importantclassof

druggablegenes.TheSilencer®SelectHumanGPCRsiRNALibraryV4targets380non-olfactoryhumanGPCRswiththreeindividualSilencer®Select

siRNAspertargetsuppliedin96-wellplates.

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Human Protease, Ion Channel, and Nuclear Hormone Receptor siRNA LibrariesFocusedsiRNAsetstohumanproteases,ionchannels,andnuclearhormonereceptorsprovideacosteffectivemeansofstudyingtheseimpor-

tantregulatorsofcellularfunction.ThesesiRNAlibrariesareeachsuppliedin96-wellplates.TheSilencer®SelectHumanProteasesiRNALibrary

V4features3siRNAspertargetfor494humanproteases.TheSilencer®SelectHumanIonChannelsiRNALibraryV4features3siRNAspertarget

for338humanionchannels.TheSilencer®SelectHumanNuclearHormoneReceptorsiRNALibraryV4includes3siRNAspertargetfor47nuclear

hormonereceptors.

To create these siRNA libraries, the Applied Biosystems bioinformatics team uses PANTHER (www.pantherdb.org) and Gene Ontology™

annotation information tocreateup-to-date targetgenesets. siRNAs targeting thecollectionare thenplated toorder (minimum0.25nmol),

givingyounotonlythemostup-to-datesiRNAlibraries,butalsothefreedomtocustomizeyourlibrarysothatitismostconvenientforyourpar-

ticularexperimentalsetup.WanttoaddsiRNAstoyourcollection?Noproblem!WecanalsoprovidesiRNAsin96-wellor384-wellplates,deliver

multiplealiquotsofeachplate,arrangesiRNAsincustomconfigurations,andprovidesiRNAsindividuallyand/orinpools.

(Ourmostpopularcollectionsarealsoavailableaspre-plated,ready-to-shiplibrarieswith0.25nmolofeachsiRNA.)

Custom siRNA libraries—the ultimate in flexibilityDoyouhaveafavoritelistofhumangenesyouwouldliketotargetwithacollectionofsiRNAs?WecanprepareacustomSilencer®SelectsiRNA

Librarytoanysetofhumangenes.Theminimumorderisonly36siRNAs.AllyouneedtosupplyisalistofgenesortranscriptsidentifiedbyNCBI

EntrezGene IDorRefSeqmRNAaccessionnumber.Withthisuser-friendlyoption,youcanspecify thesiRNA layoutoneither96-or384-well

plates.Fora96-wellplate,ourstandardformatincludesyourchoiceof0.1,0.25,1,2,or5nmolofeachsiRNA,withthreeindividualsiRNAsprovided

pertarget.Fora384-wellplate,ourstandardformatincludesyourchoiceof1,2,or5nmolofeachsiRNA,withthreeindividualsiRNAprovidedper

target.Multiplealiquots,poolingofsiRNAs,andevencustomsiRNAdesignrequestscanallbeaccomodated.

Information to accelerate discovery. AllSilencer®SelectsiRNALibraries,whethercustom,pre-defined,orpre-plated,aresuppliedwithfullsiRNA

sequenceinformation,andwhenavailable,withsiRNAvalidationdata.AlsoprovidedontheCDaccompanyingthesiRNAlibraryaregeneannota-

tioninformation,whichincludesgenesymbol,genename,aliases,NCBIEntrezGeneID,associatedRefSeqmRNAaccessionnumbers,andother

accessionnumbers.Platesareindividuallybarcodedwithuniqueidentifiers;thisinformationisalsoprovidedwiththesiRNAandgeneinformation.

Individual siRNAs for enhanced data reliabilityAllSilencer®SelectsiRNALibrariesfeaturethreeindividualsiRNAsforeachtarget.ScreeningwiththreesiRNAspergenesignificantlydecreases

both falsepositiveand falsenegative rates, increasingconfidence inRNAi screeningdata, reducing the riskofmissing importantgenes,and

decreasingtimespentfollowinguponfalsepositivehitsfromthescreen.

Silencer®SelectsiRNALibrariescontain0.25nmolofeachsiRNA,sufficientfor500transfections(at5nMsiRNA,100µLtransfectionvolume).

Preloaded96-wellplatesaresuppliedwithlyophilizedsiRNAs.Onecolumnofeach96-wellplateisleftemptytoallowfortheadditionofcontrols

atthetimeofuse.ThelibrariesaredeliveredwithfullsiRNAsequenceinformation,aswellastheexontargetedandeachtargetedgene’ssymbol,

fullname,aliases,RefSeqnumber,andEntrezGeneID,plusresultsfromanyvalidationexperimentsdoneusingtheincludedsiRNAs.

Silencer®SelectsiRNALibrariestotheseoranyotherlistofhumangenescanalsobeprovidedwith1,2,or5nmolofeachsiRNA.Customformat-

tingisavailablewiththeselargersizes.

Ifyouwouldlikeagenelist,additionalinformation,orwouldliketodiscussyourresearchwithoneofourtechnicalexperts,contactyour

localInvitrogensalesrepresentativeore-mailusatrnairesearcher@invitrogen.com.

7siRNA technologies



Silencer® Pre-designed and Validated siRNAs

Traditional, unmodified siRNAsForhuman,mouse,andrat:

→ Costeffective,unmodifiedsiRNAsdesignedforhuman,mouseandratgenes

→ Guaranteedsilencing

→ Providedwithfreesequenceinformation

Silencer® siRNAs are remarkably effective and guaranteed to silenceSilencer®Pre-designedsiRNAs—chemicallysynthesized,unmodifiedsiRNAsavailableforhuman,mouseandratgenes—aredesignedwitharig-

orouslytestedsiRNAdesignalgorithm(seeSilencer®SelectPre-designedsiRNAsonpage40forchemicallymodifiedsiRNAsdesignedwithaneven

moreeffectivealgorithm).InvitrogenguaranteesthatwhenthreeSilencer®Pre-designedsiRNAsareobtainedtothesametarget,atleasttwowill

reducetargetmRNAlevelsby70%ormore.

Silencer®ValidatedsiRNAsare individualsiRNAduplexesthathavealreadybeenverifiedexperimentallytoreducetheexpressionoftheir

individualtargetgenes.EachsiRNAwasdesignedusingthesameeffectivealgorithmusedtodesignSilencer®Pre-designedsiRNAs.However,each

onehasalsobeenfunctionallyprovenandisguaranteedtoreducetargetgeneexpressionbyatleast70%48hoursposttransfection.

High-quality synthesisInvitrogensynthesizesandpurifieseachsiRNAinstate-of-the-artfacilitiestomeetthehighestqualitystandards.Aspartofourrigorousquality

controlprocedures,weanalyzethemassofeachRNAoligonucleotidebyMALDI-TOFmassspectrometryandassessthepurityofallHPLC-purified

oligonucleotidesbyHPLC.Finally,weanalyzeeachannealedsiRNAbygelelectrophoresistoconfirmthatthestrandsannealedproperly.Theresult

ispremiumqualitysiRNAthatispurifiedandreadytouse.Seepage20foradescriptionofsiRNApuritygrades.

Silencer® siRNAs are easy to obtainInvitrogens’searchableonlinesiRNAdatabasemakesiteasytoobtaineffective,guaranteed-to-worksiRNAs.Simplyvisitwww.invitrogen.com/

siRNAtofindsiRNAsforyourhuman,mouse,orratgeneofinterest.Interestedinotherorganisms?InvitrogenprovidesCustomDesignedSilencer®

andSilencer®SelectsiRNAs(seepage49).

Order Silencer® siRNAs at www.invitrogen.com/RNAi.

Chapter references1. JacksonALetal.(2003)Nat Biotechnol 21:635–637.

2. SaxenaSetal.(2003)J Biol Chem 278:44312–44319.

3. ScacheriPCetal.(2004)Proc Natl Acad Sci U S A 101:1892–1897.

4. DuQetal.(2005)Nucleic Acids Res 33:1671–1677.

5. HornungVetal.(2005)Nat Med 11:263–270.

6. JudgeADetal.(2005)Nat Biotechnol 23:457–462.

7. SemizarovDetal.(2003)Proc Natl Acad Sci U S A 100:6347–6352.

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Chapter 8Vector-based RNAi Technologies

www.invitrogen.com

CHAPTER 8

Vector-based RNAi technologiesRNAi vectors for increased RNAi experimental optionsBLOCK-iT™RNAivectorsofferavarietyofoptionsforlong-termortransientRNAiexpression.WithBLOCK-iT™RNAi

viraldeliverysystems,RNAiexperimentscanbeperformedinthemostbiologicallyrelevantcelltyperatherthan

incelllinesthatareeasytotransfect.TheBLOCK-iT™induciblesystemspermitRNAiinitiationanddurationtobe

controlled,whiletheBLOCK-iT™PolIImiRRNAivectorslimitknockdowntospecifictissuesorcells.Thereisasuit-

ableBLOCK-iT™RNAivectorforeveryRNAiapplication.

Invitrogen has developed two distinct vector-based RNAi systems for gene knockdown experiments:

BLOCK-iT™PolIImiRRNAiExpressionVectorsandBLOCK-iT™shRNAVectors.BothPolIImiRRNAiandshRNAvector

approachesincludethecapabilityforstableandinducibleexpressionandviraldelivery.

ThepowerfulnewBLOCK-iT™PolIImiRRNAiExpressionVectorshavesignificantadvantagesovertheshort-

hairpinRNA(shRNA)vectortechnologycurrentlyusedforRNAivectorapplications(Table8.1).Thesenewvectors

include flanking and loop sequences from an endogenous miRNA that directs the excision of the engineered

miRNAfromalongerPolIItranscript(pri-miRNA).Whenpresentinthenucleus,thesevectorsefficientlyusethe

endogenous cellular machinery to process knockdown sequences that are specifically designed to have 100%

homology to the target mRNA and will result in target cleavage. In addition, the loop sequence has a unique

restrictionsite, so that itcanbe linearized toallow for trouble-freesequencing,as sequencingstandardshRNA

hairpinsissometimesachallenge.

SeeTable8.2foracomprehensiveoverviewofavailablevectorsforyourRNAiexperiments.

Table 8.1. Comparison of standard shRNA vectors and BLOCK-iT™ Pol II miR RNAi expression vectors.

BLOCK-iT™ shRNA vector technologies shRNA miR RNAi

Typicalknockdownsuccessrate† ~50% >75%

Expressiontracking No Yes

Multiple-targetknockdown No Yes

Tissue-specificexpression No Yes

Gateway®vectorcompatibilitywithmostDESTvectors No Yes

†RateatwhichconstructsreducetargetmRNAexpression>70%

8Vector-based RNAi Technologies



Table 8.2. BLOCK-iT™ vector technologies.

VectorRNAi vector technology

Transient or stable expression

Selection marker

Delivery method

Constitutive or inducible expression Reporter Kits Page

BLOCK-iT™PolIImiRRNAiexpressionvectorkits

pcDNA™6.2-GW/miR

miRRNAiTransientandstable

Blasticidin Transfection

ConstitutivePolII(CMV)Inducibleandtissue-specificoptions

None

BLOCK-iT™PolIImiRRNAiExpressionVectorKit(alsoincludedinBLOCK-iT™LentiviralPolIImiRRNAiExpressionSystem)

57

pcDNA™6.2-GW/EmGFP-miR


Blasticidin Transfection

ConstitutivePolII(CMV)Inducibleandtissue-specificoptions

CocistronicEmeraldGFP(EmGFP)

BLOCK-iT™PolIImiRRNAiExpressionVectorKitwithEmGFP(alsoincludedinBLOCK-iT™LentiviralPolIImiRRNAiExpressionSystem)

57

BLOCK-iT™PolIImiRRNAilentiviralexpressionsystems

pLenti6/V5-DEST


BlasticidinViraltransduction(ortransfection)

ConstitutivePolII(CMV)

NoneBLOCK-iT™LentiviralPolIImiRRNAiExpressionSystem(includesBLOCK-iT™PolIImiRRNAiExpressionVectorKit)

58

pLenti6/V5-DEST



ConstitutivePolII(CMV)


BLOCK-iT™LentiviralPolIImiRRNAiExpressionSystemwithEmGFP(includesBLOCK-iT™PolIImiRRNAiExpressionVectorKitwithEmGFP)

58

pLenti6.4/R4R2/V5-DEST


BlasticidinVitaltransduction(ortransfection)

Constitutive(CMVorEF-1α)orother


BLOCK-iT™LentiviralPolIImiRRNAiExpressionSystemwithEmGFP(includesBLOCK-iT™PolIImiRRNAiExpressionVectorKitwithEmGFP)

58

BLOCK-iT™shRNAentryvectors

pENTR™/U6 shRNA Transient None Transfection Constitutive None

BLOCK-iT™U6RNAiEntryVectorKit(alsoincludedintheBLOCK-iT™RNAiLentiviralExpressionSystemandtheBLOCK-iT™AdenoviralRNAiExpressionSystem)

66

pENTR™/H1/TO shRNATransientandstable

Zeocin™ TransfectionInducibleorconstitutive

None

BLOCK-iT™InducibleH1RNAiEntryVectorKit(alsoincludedintheBLOCK-iT™InducibleH1LentiviralRNAiSystem)

66

BLOCK-iT™shRNAdestinationvectors

pLenti6/BLOCK-iT™-DEST

shRNATransientandstable


Constitutive None

BLOCK-iT™RNAiLentiviralExpressionSystemBLOCK-iT™LentiviralRNAiGateway®VectorKit

69



Zeocin™Viraltransduction(ortransfection)

Inducibleorconstitutive

None

BLOCK-iT™InducibleH1LentiviralRNAiSystemBLOCK-iT™LentiviralRNAiZeoGateway®VectorKit

71

pLenti6/TR(forexpressionoftetracyclinerepressoronly)

shRNAormiRRNAi

Transientandstable


Constitutive NonepLenti6/TRVectorKitBLOCK-iT™InducibleH1LentiviralRNAiSystem

70

pAd/BLOCK-iT™-DEST

shRNA Transient NoneViraltransduction(ortransfection)

Inducibleorconstitutive

NoneBLOCK-iT™AdenoviralRNAiExpressionSystempAd/BLOCK-iT™-DESTRNAiGateway®Vector

69

pBLOCK-iT™6-DEST


Blasticidin Transfection Constitutive None pBLOCK-iT™6-DEST 69

pBLOCK-iT™3-DEST


Geneticin® TransfectionInducibleorconstitutive

None pBLOCK-iT™3-DEST 71

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Table 8.2. BLOCK-iT™ vector technologies, continued.

VectorRNAi vector technology

Transient or stable expression

Selection marker

Delivery method

Constitutive or inducible expression Reporter Kits Page

SelectedGateway®DESTvectorscompatiblewithmiRRNAivectortechnology*

pT-REx™-DEST30


Geneticin® TransfectionInducible(CMV)

CocistronicEmGFP

Nokitavailable.VectorCat.No.12301-016.NeedstobeusedinaT-REx™celllineorincellsthatexpresstheTetrepressorprotein.

62

pLenti4/TO/V5-DEST


Zeocin™Viraltransduction(ortransfection)

Inducible(CMV)

CocistronicEmGFPornone†

ViraPower™T-REx™LentiviralExpressionSystem.NeedstobeusedinaT-REx™celllineorincellsthatexpresstheTetrepres-sorprotein.

62

pEF-DEST51 miRRNAiTransientandstable

Blasticidin TransfectionConstitutive(EF-1α)

CocistronicEmGFPornone†

Nokitavailable.VectorCat.No.12285-011 62

pDEST™R4-R3 miRRNAi Transient None Transfection YourchoiceCocistronicEmGFPornone†

MultiSiteGateway®Three-FragmentVectorConstructionKit

62

pLenti6/R4R2/V5-DEST



YourchoiceCocistronicEmGFPornone†

ViraPower™PromoterlessLentiviralGateway®ExpressionSystemwithMultiSiteGateway®Technology

62

pLenti6/TR(forexpressionoftetracyclinerepressoronly)

shRNAormiRRNAi

Transientandstable


Constitutive None pLenti6/TRVectorKit 62

pcDNA™6/TR(forexpressionoftetracyclinerepressoronly)

shRNAormiRRNAi

Transientandstable

Blasticidin TransfectionConstitutive(CMV)

None T-REx™CoreKitorT-REx™CompleteKit 62

pSCREEN-iT™/lacZ-DEST

shRNAormiRRNAi

Transient None Transfection Constitutive lacZ

BLOCK-iT™RNAiTargetScreeningSystem;BLOCK-iT™RNAiTargetScreeningKit;pSCREEN-iT™/lacZ-DESTGateway®VectorKit

64

Ready-to-usevectors:clonedandsequenceverified

miRRNAiorshRNA

Transientandstable

Blasticidin,Zeocin™,orGeneticin®

Transfectionorviraltransduction

Inducibleorconstituitive

CustomRNAiServices 93

*Additionalcomponents,suchasClonase™enzymemixesandGateway®donorvectors,areneededtotransferthemiRRNAicassetteintotheseDESTvectors.TheadditionalcomponentsareincludedintheBLOCK-iT™LentiviralPolIImiRRNAiExpressionSystembutarenotincludedinBLOCK-iT™PolIImiRRNAiexpressionvectorkitsandmustbepurchasedseparatelytotransferthemiRRNAiexpressioncassettetoalternativeDESTvectors.

†CocistronicEmGFPreportercanbetransferredfromtheoriginalBLOCK-iT™PolIImiRRNAiexpressionvector.




How vector-mediated RNAi works

BothmiRRNAiandshRNAvectorsystemstakeadvantageoftheendogenousRNAipathwayfoundinallanimalcells.ComparedtoshRNAvectors,

themiRRNAivectorsystems,byusingartificialmicroRNAs(miRNAs),utilizemoreofthecomponentsoftheendogenousmachinery,resultingin

moreefficientprocessingofexpressedRNAhairpins(Figure8.1).

BLOCK-iT™Pol IImiRRNAiexpressionvectorkitsutilize theendogenousmiRNApathway.ArtificialmiRNAsexpressed fromthepcDNA™

6.2-GW/EmGFP-miRExpressionVectoraretranscribedbyRNAPolymeraseII,whichenablescocistronicexpressionofEmeraldGreenFluorescent

Protein(EmGFP)andmultiplemiRNAhairpinsonthesametranscript.TheprimarymiRNA(pri-miRNA)transcriptcontainstheEmGFPsequence

onthe5´end,followedbyoneormoreprecursormiRNAs(pre-miRNAs).TheRNasetypeIIIenzymeDrosharecognizestheflankingsequencesof

thepre-miRNAsandexcisesthemfromthepri-miRNAtranscript.EachprecursormiRNAisthenactivelytransportedoutofthenucleusbyXpo-5.

Once in the cytoplasm, the pre-miRNA hairpins are processed further by Dicer, which converts them into miRNAs. Finally, the miRNAs

unwind,loadintotheRNA-inducedsilencingcomplex(RISC),andhybridizewiththeirmRNAtarget.WhilemostendogenousmammalianmiRNAs

donotperfectlycomplementthetargetmRNAsequenceandthusresultintranslationalinhibition,theartificiallydesignedmiRNAsusedinthis

systemare100%homologoustothetargetmRNAsequenceandresultintargetcleavage.

TheshorthairpinRNA(shRNA)vectorscontainanRNAPolymeraseIII (Pol III)promoter(H1orU6)fornuclearexpressionofshRNAs[1–5]

Exportin-5activelyexportsshRNAtothecytoplasm[6,7],where it is recognizedandcleavedbytheRNase IIIenzymeDicer toproduceshort

interferingRNA(siRNA)[3].

i

miRNA has 100% homology to mRNA,which results in target mRNA cleavage

Drosha

AAAAAA

Primary miRNA(pri-miRNA)

Precursor miRNA(pre-miRNA)

Dicer

Nuclearpore

Xpo-5

miRNA

Unwinding

RISC

Nucleus Cytoplasm

EmGFP

Pol II mRNA transcription

m7Gppp

P CMV

SV40 pA

TK pA

Blastic

idin

Spectinom

ycin

f1 oriSV40 ori

EM7

pUC ori


5,699 bp

Figure 8.1. Expression of miR RNAi sequences using the BLOCK-iT™ Pol II miR RNAi expression vectors.

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BLOCK-iT™ Pol II miR RNAi expression vectors—versatile Pol II vector-based RNAi technology

BLOCK-iT™PolIImiRRNAiexpressionvectorscombinetheadvantagesoftraditionalRNAivectors—stableexpressionandtheabilitytouseviral

delivery—withcapabilitiesfortissue-specificexpressionandmultiple-targetknockdownfromthesametranscript.ThepcDNA™6.2-GW/miRand

pcDNA™6.2-GW/EmGFP-miRvectors(Figure8.2),includedintheBLOCK-iT™PolIImiRRNAiexpressionvectorkitsandtheBLOCK-iT™Lentiviral

PolIImiRRNAiExpressionSystem,aredesignedforexpressingartificialmiRNAsthathavebeenengineeredtohave100%homologytoatarget

sequenceandwillresultinitscleavage.TheBLOCK-iT™PolIImiRRNAiExpressionVectortechnologyoffersmanybenefits:

→ Over75%knockdownsuccess—screenfewerclonesbecausedesignpredictabilityishigherthanthatofothervector-basedRNAimethods

→ Easyexpressiontracking—assessknockdownoftargetmiRNAsimplyandreliablywithcocistronicexpressionwithEmGFP

→ Multiple-targetknockdown—knockdownmultipletargetssimultaneouslyorgeneratesyntheticphenotypes

→ Gateway®vectorcompatibility—useawideselectionofInvitrogen™destinationvectors,includinglentiviralvectors,forstableexpressionin

anycelltype,suchasprimaryandnondividingcells

→ Tissue-specificexperimentaloptions—selectfromavarietyofdestinationvectorsorMultiSiteGateway®vectorapplicationstoadddifferent

promotersforcellularapplicationsorin vivo knockdown

→ Inducible expression—regulation of the RNAi response permits the study of changes over time and loss-of-function experiments, and

providesanexcellentcontrolsystemtomeasurephenotypicchangesduringrecoveryofgenefunction

→ BLOCK-iT™miRRNAiSelect—targetthemajorityofannotatedhuman,mouse,andratgeneswithpredesignedBLOCK-iT™miRRNAiSelect

hairpins,readytoannealandcloneintoeitheroftheBLOCK-iT™PolIImiRRNAiexpressionvectors

P CMV

SV40 pA

TK pA

Blastic

idin

Spectinom

ycin

f1 oriSV40 ori

EM7

pUC ori


5,699 bp

attB1 EmGFP 5´ miR�anking region ACGA attB23´ miR

�anking regionCAGG

P CMV

SV40 pA

TK pA

Blastic

idin

Spectinom

ycin

f1 oriSV40 ori

EM7

pUC ori

pcDNA™6.2-GW/miR

4,944 bp

attB1 5´ miR�anking region ACGA attB23´ miR

�anking regionCAGG

Figure 8.2. The BLOCK-iT™ Pol II miR RNAi expression vectors. ThepcDNA™6.2-GW/miRvector isdrivenbytheCMVpromoter,hastheblasticidinresistance-marker,andisavailablewithorwithoutcocistronicEmGFPexpressionasareporter.

NOTE: Find information on miR RNAi expression vectors online at www.invitrogen.com/mir.




Combine BLOCK-iT™ vector kits with lentivirus for stable, long-term expression

BLOCK-iT™andBLOCK-iT™HiPerform™LentiviralPolIImiRRNAiExpressionSystemscombinealloftheadvantagesoftheBLOCK-iT™PolIImiRRNAi

ExpressionVectorKitwithViraPower™LentiviralExpressionVectors(Figure8.3),enablingstabledeliveryofengineeredmiRNAsintonondividing,

primary,andhard-to-transfectcells.Forexample,ViraPower™LentiviralExpressionVectorshavebeenusedtodelivermiRRNAisequencesinto

ratprimarycorticalneuronstoknockdownmicrotubule-associatedprotein2(Figure8.4).ThesevectorsdelivermiRNAsequencesintoHT1080

andHeLacellstoknockdownlaminA/CorlacZ (Figure8.5),establishinglong-term,stableexpressionofmiRNAsinthesecellmodelsystems.

TheBLOCK-iT™HiPerform™LentiviralPolIImiRRNAiExpressionSystemwithEmGFPisthemostpowerfulandflexibleRNAivectoroffered

todate.Thistechnologycombineshightitersandmaximumexpression.Theabilitytoincorporatecustompromotersmakesthesystemsuitable

forin vivo applications.

TheHiPerform™vectorcontainsanmRNA-stabilizingsequence(WPRE)andanuclearimportsequence(cPPT)thatcangenerateupto5-fold

highervirustitersandEmGFPexpressionlevelsinmanycelllines.Additionally,MultiSitetechnologyallowsyoutoexpresstheEmGFP/miRRNAi

cassettefromCMV,EF-1α,yourowntissue-specificpromoter,oranotherappropriatein vivopromoter:

→ Achieveupto5xhighertiters (measuredbyGFP),allowingmorecells tobetransducedorhighermultiplicitiesof infection(MOIs) tobe

employed

→ Incorporateyourowntissue-specificoranotherappropriatein vivopromoter

→ TrackmiRNAexpressionthroughcocistronicexpressionwithEmGFP

→ KnockdownmorethanonegenesimultaneouslythroughexpressionofmultiplemiRNAsfromasingletranscript

Figure 8.3. pLenti6/V5-DEST vector. A.TheBLOCK-iT™LentiviralPolIImiRRNAiExpressionSystemsutilizethepLenti6/V5-DESTvectorforconstitutiveexpres-sion from the CMV immediate-early promoter, and the blasticidin selection marker for compatibility with a tetracycline-regulated inducible system.B.Imagestaken4daysfollowingtransductionofGripTite™293MSRcellsatanMOIof3withlentiviralparticlesgeneratedusingtheindicatedvectors.

pLenti6.4/CMV orEF-1α/MSGW/EmGFP-miR

8,875 bp

PR

SV/5

´ LTR

RRE

∆E3/3´ L

TRB

last

icid

in

SV40 pA

Ampicillin

pUC

ori

PPGK

EM7

attB1 attB2attB4 EmGFP 5´ miR�anking region

3´ miR�anking regionPCMV

cPPT WPRE

A

pLenti6-GW/EmGFP-miR-negpLenti6.4/CMV/V5- MSGW/EmGFP-miR-negB

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No treatment

Lenti-miR-Neg

Lenti-miR-MAP2A

Figure 8.4. Target gene knockdown in primary neurons. Unlikestandardretroviruses, lentivirusescantransducenondividingcells,suchastheseratprimarycorticalneurons.Here,microtubule-associatedprotein2(MAP2A)wastargeted,anditsexpressionwasdramaticallydecreased.Notreatment(AandB),Lenti-miR-Neg(CandD),andLenti-miR-MAP2(EandF)werestainedforanti-MAP2andDAPI.ImageswereacquiredwithaNikonE800microscopeandtheOmegaXF-32filtercubefortheAF555channelandtheOmegaXF136-2filtercubeforDAPI.PanelsA,C,andE:20xmagnification.PanelsB,D,andF:40xmagnification.Thereisavisibledifferencebetweenthetransfectedcontrolandthelenticontrolvs.lentiviraldeliveryofanRNAiknockdownreagent.

Mag

icM

ark™

Lam

in

Day33

80 kDa Lamin A/C

β-actin

60 kDa

50 kDa

40 kDa

Day48

Day64

Day85

Day99

lacZ

Lam

in

lacZ

Lam

in

lacZ

Lam

in

lacZ

Lam

in

lacZ

Figure 8.5. Stable lentiviral transduction of miRNA expression cassettes. WesternblotanalysisofHeLacellpopulationsstablytransduced(MOI=20)for33to99dayswithlaminorlacZ miRNA–expressinglentiviralparticles.Theblotwasprobedwithanti–laminA/C(tophalf)oranti–β-actin(bottomhalf)antibodies.




Reliable tracking of the RNAi cassetteDeterminingwhichcellsareexpressingtheartificialmiRNAofinterestisstraightforwardwithcocistronicexpressionofEmGFP.ThepcDNA™6.2-GW/

EmGFP-miRvector(includedintheBLOCK-iT™PolIImiRRNAiExpressionVectorKitwithEmGFPortheBLOCK-iT™HiPerform™LentiviralPolIImiR

RNAiExpressionSystemwithEmGFP)expressesEmGFPcocistronicallywiththemiRNAofinterest,sotheexpressionofEmGFPcorrelatesnearly

100%withtheexpressionofthemiRNA(Figure8.6),allowingmiRNAexpressiontobetrackedinanycelltype.

Figure 8.6. 100% correlation of EmGFP and miRNA expression. (A)MapoftheBLOCK-iT™PolIImiRRNAiexpressioncassetteshowsEmGFPbetweenDraIrestric-tionsites foreasy removal. (B)Cellswere transfectedwithpcDNA™6.2-GW/EmGFP-miR (directedagainst lamin)andLipofectamine®2000TransfectionReagent,atanexpected50%efficiency,todemonstratethe100%trackingofEmeraldGreenFluorescentProtein(EmGFP)andmiRNAexpression.After48hrthecellswerestainedwithHoechstnuclearstain,whichstainsallcells(leftpanel),andaredlaminstain(centerpanel),andmonitoredforEmGFPexpression.Approximatelyhalfofthecellshighlyexpressedthelaminprotein(compareleftandcenterpanels),butred-stainedcellswerenotexpressingEmGFP,andEmGFP-expressingcellswerenotexpressinglamin(rightpanel).ThisdemonstratesthecocistronicexpressionofEmGFPandofthemiRNAthatgreatlyreduceslaminexpression.

miRNA

Web-designed insert

EmGFP

att att

A

Lamin A/CHoechst EmGFP + Lamin A/C

B

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Knockdown of multiple targets for experimental flexibilityBLOCK-iT™PolIImiRRNAiexpressionvectorsaredesignedfortheeffectiveknockdownofmorethanonetargetinasingleexperiment.ThePolII

promoterenablescocistronicexpressionofmultiplemiRNAs,allowingknockdownofmultipletargetsfromasingleconstruct(Figure8.7).Witha

simplerestrictionenzymeprocedure,twoormoremiRNAsequencescanbelinkedinanyorder.Thisprocessisidealforknockdownofmorethan

onepathwaycomponentorsplicevariant,orforusingknockdowntocreatesyntheticphenotypes.

Figure 8.7. Knock down multiple targets with BLOCK-iT™ Pol II miR RNAi expression vectors. (A)Exampleofrestrictiondigestion/ligationschemeforcombiningmiRNAsfromdifferentvectors.SalI(S),BamHI(B),BglII(Bg),andXhoIsites(X)aroundthemiRNAflankingregions(bars)areindicated.BycloningtheBamHI–XhoIfragmentcontainingmiRNA1intotheBglI–XhoIfragmentofthevectorcontainingmiRNA2,adual-miRNAplasmidiscreated.Theoriginalrestrictionsitepattern is recreated (restrictionsitesbetweenthemiRNAsaredestroyed)andadditionalmiRNAscanbeadded in thesamemanner.Alternatively,miRNAscanbeaddedinfrontofmiRNA1bycombiningSalI–BglIIandSalI–BamHIfragments.(B)CotransfectionofluciferaseandlacZ reporterplasmidswithsingle-ordual-miRNAvectorswiththeindicatedinserts.Luciferaseandβ-galactosidaseactivitiesarenormalizedtothesingle(neg)ordual(neg/neg)miRNAnegativecontrolinserts,whichformapre-miRNAbutarenotpredictedtotargetanyknownvertebrategenes.Knockdownisslightlyattenuatedinthedual-miRNAvectorsbutremainsverypotentat≥90%.

S B Bg X

S B Bg X

S B Bg X

1

2

2 1

A

β-gal

luc

NegNeg

NeglacZ

lucNeg

luclacZ

Neg—

Single-miRNA plasmid

lacZ—

luc—

miR-1miR-2

Rep

orte

r ac

tivity

(% o

f con

trol

) 140

100

40

120

60

80

20

0

miRNA(s)

Dual-miRNA plasmid

B




Gateway® vector compatibility for expanded experimental options

Gateway®technologyisafastandefficientwaytotransfermiRRNAicassettesintoavarietyofvectorsbyhomologousrecombination.Gateway®

recombinationeliminatestediousandtime-consumingsubcloningprocedures,andtheresultingexpressioncassettescanbeeasilymovedinto

anyoftheGateway®DESTvectors.SeeTable8.3foralistingofGateway®destination(DEST)vectorsthathavebeenfunctionallytestedforcom-

patibilitywiththeBLOCK-iT™PolIImiRRNAiVectorKits.

WiththeBLOCK-iT™Pol IImiRRNAiExpressionVectorKits,miRNAsarecloneddirectly intoGateway®expressionvectors,asopposedto

Gateway®entryvectors.Asaresult,therearetwokeydifferencesbetweenthepcDNA™6.2-GW/miRandpcDNA™6.2-GW/EmGFP-miRexpression

vectorsandtypicalGateway®entryvectors:

1. ThemiRNAexpressionvectorsincludethecytomegalovirus(CMV)promoter.AftermiRNAsequencesarecloned,theyareimmediatelyready

fortransfectionandmiRNAexpression.

2. attB sites flank the miRNA (and EmGFP sequences if using pcDNA™6.2-GW/EmGFP-miR). For expression in different DEST vectors, the

insertsmustfirstbetransferredtoapDONR™(donor)vectorandthentoaDESTvectorofchoicebyadualClonase®enzymereaction.The

pDONR™221vector,BPandLRClonase®IIenzymemixes,andpLenti6/V5-DESTvectorareincludedintheBLOCK-iT™LentiviralPolIImiR

RNAiExpressionSystem.

Pol II promoter versatility allows tissue-specific expressionMostshRNAvectorsaredrivenbyPolIIIpromoters,significantlylimitingthetypesofpromotersthatcanbeusedinRNAiexperimentsandmaking

tissue-specificexpressioninanin vivo systemimpossible.OthervectorapproachesusespeciallymodifiedPolIIpromoters,whichcannotbeeasily

exchangedforotherPolIIpromoters.TheBLOCK-iT™PolIImiRRNAiExpressionVectorsincludetheCMVimmediate-earlyPolIIpromoterandare

compatiblewithvirtuallyanyotherPolIIpromoter(Figure8.8),providingaflexiblesystemtoregulateknockdownortousepromotersthatare

activeinspecifictissuesforin vivo studies.

Inducible expression for control over your experimentThenewBLOCK-iT™ InduciblePol IImiRRNAiExpressionVectorKitwithEmGFPenablescustomers to regulateRNAiexperiments (seeFigure

8.8B).NowyoucanobservechangesovertimebycontrollingtheinitiationoftheRNAiresponsewithaninduciblesystem.Thekitcontainsthe

pT-REx-DEST30Gateway®vectorwhich,aftersimplecloningandshuttlingtechniques,producesamiRRNAiexpressionvectorsuitableforinduc-

ibleknockdown.ThepT-REx-DEST30Gateway®vectorcontainstheCMVpromoterwithtwocopiesofthetetracyclineoperator(TetO2)sequence,

allowinghigh-levelandregulatedexpression(Figure8.9).Thispermitsthestudyof lossoffunctioninastablytransfectedcell lineevenifthe

geneofinterestisessential.Also,inductionofmiRRNAiexpressioncanbehaltedsophenotypicchangescanbemeasuredduringrecoveryof

genefunction.

Table 8.3. Compatibility of Gateway® destination vectors.

Promoter or system Compatibility Cat. No.

ViraPower™lentiviralvectors,includingMultiSiteGateway®technology

Bench-testedwithpLenti6/V5-DESTandpLenti6/UbC/V5-DEST

V496-10V499-10

EF-1αpromoter Bench-testedwithpEF-DEST51 12285-011

T-REx™vector Bench-testedwithpT-REx™DEST30 12301-016

Flp-In™cellline Bench-testedwithpEF5/FRT/V5-DEST V6020-20

N-terminalreportertags Bench-testedwithpcDNA™6.2/N-YFP/DEST V358-20

MultiSiteGateway®technologyBench-testedwithpDEST™/R4-R3usingthymidinekinase(TK)poly(A)3´elementandvarious5´promoterelements

12537-023

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β-gal

luc

Neg AAK1 lacZ luc2AAK1

300 ng CMV

lacZ luc2Neg

Rep

orte

r en

zym

e ac

tivity

(% o

f neg

con

trol

s)

100

40

120

140

60

80

20

0

300 ng EF-1α

A

luc unind.

luc ind.

β-gal unind.

β-gal ind.

lacZ luc2AAK1Neg

Rep

orte

r en

zym

e ac

tivity

(% o

f neg

con

trol

s)

120

100

80

60

40

20

0

30 ng CMV/TO

BR

epor

ter

activ

ity (%

of c

ontr

ol)

(1 x

1,0

00 R

FU)

luc2lacZNeg

200

0

400

600

800

1,000

1,200

β-gal

Rep

orte

r ac

tivity

(% o

f con

trol

)(R

FU)

luc2lacZNeg0

5,000

10,000

15,000

20,000

25,000

luc

C

Figure 8.8. BLOCK-iT™ Pol II miR RNAi Expression Vectors are compatible with multiple promoters. (A)NormalizedreporteractivitiesfromlysatesofGripTite™293cellscotransfectedwith100ngeachof luciferaseand lacZ reporterplasmidsand300ng/wellofeitherpcDNA™6.2-GW/EmGFP-miR(CMV)orpEF-GW/EmGFP-miR (EF-1α)expressionvectors. (B)KnockdownofpSCREEN-iT™/lacZ-AAK1vectorand luciferase reporters inT-REx™293cellswithandwithoutinductionwith1μg/mLtetracyclineat30ngofpT-REx™-GW/EmGFP-miR(CMV/TO)plasmidperwell.(C)KnockdownofcotransfectedlacZ andluciferasereportergenesinHepG2cellsbyMultiSiteGateway®EmGFP-miRconstructswiththeanti-trypsinpromoterasthe5´elementandthepoly(A)signalfromtheHSVthymidinekinasegeneasthe3´element.

Repressed state

Derepressed state

H1 TetO2 TATA TetO2 miR RNAi

H1 TetO2 TATA TetO2 miR RNAi

Figure 8.9. Inducible RNAi via tetracycline regulation of the CMV/TO Pol II promoter. Whenthetetracyclinerepressor(TR)proteinispresentinthecell,ittightlybindsthetwoTetO2siteswithintheH1promoter,essentiallyblockinginitiationofshRNAtranscription.Whentetracyclineisaddedtotheculturemedium,itbindsto,andchanges,theconformationoftheTRprotein,causingtheTRproteintoreleasefromthetwoTetO2sites,allowingtranscriptiontooccur.




BLOCK-iT™ miR RNAi Select

NowitiseasierthanevertoharnessthepoweroftheBLOCK-iT™PolIImiRRNAiExpressionVectorswithpredesignedBLOCK-iT™miRRNAiSelect

hairpins,whichtargetthemajorityofthehuman,rat,andmousegenesintheRefSeqdatabase.TheBLOCK-iT™miRRNAihairpins,designedin

silicousingtheaward-winningBLOCK-iT™RNAiDesigner,areprovidedasDNAoligosthatarereadytoannealandcloneintooneoftheBLOCK-iT™

PolIImiRRNAiExpressionVectors.Ready-to-orderBLOCK-iT™miRRNAiSelecthairpinsprovidesignificantadvantages:

→ EasyaccesstoBLOCK-iT™PolIImiRRNAiExpressionVectortechnology,whichcombinesthehighdesignsuccessrateofartificialmiRNAs

withtissue-specific,inducible,multiplegene–targeting,andGFPreporteroptions

→ EffortlessorderingusingtheBLOCK-iT™RNAiExpresssearchengine

→ AllowsmaximumreductionoftduetocombinedpowerofBLASTandtheadvancedSmith-Watermanalignmentspecificitysearch

→ FourBLOCK-iT™miRRNAiSelecthairpindesignsdirectedtononoverlappingregionsofthetargetmRNAsequence.

WithBLOCK-iT™miRRNAiSelect4Sets,youcanavoidtheworkofdesigningRNAihairpinsequencesin-house.

Product Quantity Cat. No. miR RNAi vector kits

BLOCK-iT™PolIImiRRNAiExpressionVectorKit 20rxns K4935-00

BLOCK-iT™PolIImiRRNAiExpressionVectorKitWithEmGFP 20rxns K4936-00

BLOCK-iT™LentiviralPolIImiRRNAiExpressionSystem 20rxns K4937-00

BLOCK-iT™LentiviralPolIImiRRNAiExpressionSystemWithEmGFP 20rxns K4938-00

BLOCK-iT™PolIImiR-lacZValidatedmiRNAControlVector 10µg V49350-00

BLOCK-iT™PolIImiR-lucValidatedmiRNAControlVector 10µg V49351-00

BLOCK-iT™PolIImiR-LMNAValidatedmiRNAControlVector 10µg V49352-00

BLOCK-iT™miRRNAiSelect 1hairpin †

BLOCK-iT™miRRNAiSelect4Sets* 4hairpins †

BLOCK-iT™HiPerform™LentiviralPolIImiRRNAiExpressionSystemWithEmGFP 20rxns K4934-00

BLOCK-iT™InduciblePolIImiRRNAiExpressionVectorKitWithEmGFP 20rxns K4939-00

Recommended destination vector kits

Gateway®pT-REx™-DEST30Vector 6µg 12301-016

ViraPower™T-REx™LentiviralExpressionSystem 20rxns K4965-00

Gateway®pEF-DEST51Vector 6µg 12285-011

MultiSiteGateway®Three-FragmentVectorConstructionKit 20rxns 12537-023

ViraPower™PromoterlessLentiviralGateway®ExpressionSystemwithMultiSiteGateway®Technology 20rxns K5910-00

*Gotowww.invitrogen.com/rnaicentralfordetailsonthe100%performanceguarantee.

†Visitwww.invitrogen.com/rnaiexpressfororderinginformationonthelatestBLOCK-iT™miRRNAiSelect,BLOCK-iT™miRRNAiSelect4Sets,andBLOCK-iT™PolIImiRValidatedmiRNAVectorDuoPaksthatareavailable.

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Guaranteed results with BLOCK-iT™ miR RNAi Select 4 Sets

ThepurchaseofaBLOCK-iT™miRRNAiSelect4Setincludes100%guaranteedresults:twoofthefourdesignswillresultinatleast70%transcript

knockdown,givenatransfectionefficiencyofatleast80%.If,undertheseconditions,threeormoreofthedesignsfailtoknockdownthetarget

mRNAbyatleast70%,Invitrogenwilldesignandshiptwoadditionaltarget-specificBLOCK-iT™miRRNAihairpinsatnoexpense.*

*PleasecontactInvitrogenTechnicalServicestotakeadvantageofthisguarantee.Bepreparedtofaxoremailyourorderreferencenumber,BLOCK-iT™miRRNAiSelectsequences,anddatashowingtransfectionefficiencyandlevelofknockdown.Thisguaranteecanbeusedonlyoncepertargetgene.

WithBLOCK-iT™miRRNAiSelect4Sets,youcanavoidtheworkofdesigningRNAihairpinsequencesyourself.Thedevelopmentofthesemol-

eculesbeginswithidentificationoftheentirehuman,rat,andmousetranscriptsintheUnigenedatabase.Invitrogenthenselectsexceptionally

effectivein silico–designedsequencesbyafour-stepprocess:

→ BLASTsearchtoremovesequenceswithinthetargetmRNAthatarehomologoustoothermRNAsequenceswithinthetargetspecies

→ BLOCK-iT™miRRNAiSelecthairpindesignusingtheBLOCK-iT™RNAiDesigner,whichincludesbothstandardandproprietaryrulesbased

ontestingthousandsofRNAduplexandRNAivectorsequences

→ Smith-Watermanmismatchalignmenttorigorouslyscreensequencesforpotentialoff-targeteffects

→ SelectionofthetopfourartificialmiRNAhairpindesignstargetingnonoverlappingregionsinallmajorsplicevariantsfoundinRefSeqfor

thetargetmRNAsequence;thesedesignsareofferedindependentlyasBLOCK-iT™miRRNAiSelecthairpinsorasacompleteBLOCK-iT™

miRRNAiSelect4Set




BLOCK-iT™ RNAi Entry Vector Kits

pENTR™ vectors for fast cloning and expression of shRNA cassettesTheBLOCK-iT™InducibleH1andBLOCK-iT™U6RNAiEntryVectors(whichcontainthepENTR™/H1/TOandpENTR™/U6vectors,respectively)pro-

videasimpleandstreamlinedapproachforgeneratinganRNAicassettethatconsistsofaninducibleorconstitutivePolIIIpromoter,theshRNA

sequence,andthePolIIIterminationsequences.Thefirststepforusingthistechnologyistodesignadouble-strandedDNAoligonucleotidewith

asense-loop-antisensesequenceagainstthetargetgeneofinterest.ThissequenceencodingtheshRNAcanbereadilydesignedusingtheonline

BLOCK-iT™RNAiDesignerfoundatRNAiCentral(www.invitrogen.com/rnai).

ClonetheannealedshRNAoligosequence intothepENTR™vectorwithaconvenient5-minutecloningreaction (Figure8.10).Oncethe

shRNAcassettehasbeengeneratedinoneoftheentryvectors,thevectorcanbedelivereddirectlyintomammaliancellsforknockdownstudies,

ortheshRNAcassettecanbetransferredintoanotherBLOCK-iT™DESTvectorusingGateway®technology.

Double-stranded oligo

4–11 nt loop19–22 nt sense (or antisense)target sequence

19–29 nt antisense (or sense)target sequence

Reverse complement of top strand

AAAA5´ CACCG

C

Easy 5 min cloning into thelinearized pENTR™/U6 vector

Easy 5 min cloning into thelinearized pENTR™/H1/TO vector

Double-stranded oligo

4–11 nt loop19–22 nt sense (or antisense)target sequence

19–29 nt antisense (or sense)target sequence

Reverse complement of top strand

AAAA5´ CACCG

C

pENTR™/U62.9 kb

U6 promoter Pol III termGTGGTTTT

PH1/TO Pol III termGTGGTTTT

pENTR™/H1/TO3.9 kb

T2

T1

SV

40 p

A

Zeo

cin

™

EM

7 P SV40 a

ttL1 attL2

pUC orgin

Kan

amyc

in

T2

T1

attL1 attL2

pUC

ori Kanamycin

Figure 8.10. Easy shRNA cloning into inducible and constitutive BLOCK-iT™ entry vectors. AvectorexpressingtheshRNAisgeneratedbydesigningashortdouble-strandedDNAoligocontainingasense-loop-antisensesequenceagainstthetarget.Thesequencehas4-nucleotideoverhangsontheendsthatcanbeligated into thepENTR™/H1/TOorpENTR™/U6entryvectorviaabriefbenchtop reaction.Theentryvectorcontainseither theH1promoterwith twotetracyclineoperatorsitesflankingtheTATAregion,ortheU6PolIIItypepromoterandthePolIIIterminatorsequence.Cloningthedouble-strandedoligosequenceintothevectorcreatesanRNAicassettethatexpressestheshRNA.

NOTE : Design your shRNA oligos online at www.invitrogen.com/rnai.

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Inducibility with shRNA vectorsTheBLOCK-iT™InducibleH1EntryVectorKitconferstheabilityfortightlyregulatedinducibleRNAiinanymammaliancelltype.ThepENTR™/H1/

TOvector,includedinthekit,carriesanH1/TOPolIIIpromoterthatexhibitssuperiorregulationcomparedtoprecursorversions,whichcontained

only one TetO2 site in either position. Upon addition of tetracycline, shRNA is expressed, leading to effective knockdown of the target gene

(Figure8.11).TightcontrolovershRNAexpressionpermitsavarietyofexperimentalconditionstobeexamined:

→ ObservechangesovertimebycontrollingtheinitiationoftheRNAiresponse

→ Studylossoffunctioninastablytransfectedcellline,evenifthegeneofinterestisrequiredforcellviabilityorproliferation

→ TerminateinductionofshRNAexpressiontoobservephenotypicchangesduringrecoveryofthefunctionofthetargetgene

NOTE: Find information on shRNA vectors online at www.invitrogen.com/shrna.

+–+–+–+–1 µg/mL Tet:

Luci

fera

se a

ctiv

ity

16,000

14,000

10,000

12,000

8,000

6,000

4,000

2,000

0

Transfection condition

Untransfected lacZ shRNA lacZ shRNAluc shRNA Lamin A/C shRNA

+–1 µg/mL Tet:

Luci

fera

se a

ctiv

ity

7,000

5,000

6,000

4,000

3,000

2,000

1,000

0

Transfection condition

Untransfected Carrier plasmid luc shRNA Lamin A/C shRNA

+– +– +–+–

Figure 8.11. Tightly regulated induction of gene knockdown from the pENTR™/H1/TO vector. T-REx™-CHOcells(leftpanel)orapolyclonalpopulationofHeLacellsstablytransducedwithlentiviralparticlesexpressingtheTRprotein(rightpanel)werecotransfectedwithlacZ andtheluciferase(luc)reportergeneplusH1/TOentryclonesinduciblyexpressinglacZ-,luc-,orlaminA/C-directedshRNAs.FortheHeLacells,anonspecificcarrierplasmidwasincludedasacontrol.After3hr,mediumwasreplacedwithfreshmedium,withorwithout1μg/mLtetracycline(Tet)asindicated.Cellswerelysed48hrposttransfectionandassayedforluciferaseactivity.




Lentiviral and adenoviral RNAi vectors

Choose any cell type for RNAiFormanydiseasemodels,themostappropriatecelltypes,suchasimmunesystemorprimarycells,arenotamenabletotransfection.Viraldelivery

ofRNAivectorsisapowerfulalternativetotransfectionforthesecelltypes,aswellasforin vivo applications.Toaccuratelydeterminetheefficacy

ofknockdownfromanshRNA/miRRNAimoleculeinapopulationofcells,itiscriticaltodelivertheshRNA/miRRNAimoleculetoasmanycellsas

possible.Otherwise,whenknockdownismeasuredbyquantitativereal-timePCR(qRT-PCR)orwesternblotanalysis,thebackgroundofmRNAor

proteininnontransfectedcellswillmaketheknockdownappearlesseffectivethanitactuallyis.Viraldeliverycanbethebestoptioninvirtually

anymammaliancelltype,includinghard-to-transfect,primary,andevennondividingcells.Conveniently,lentiviraldeliverysystemsareavailable

forbothshRNAandmiRRNAivectors,andanadenoviraldeliverysystemisavailableforshRNAvectors(Table8.4).

TheprocedureforusingbothRNAiviralsystems(Figure8.12):

1. Clonethedouble-strandedDNAoligoencodinganshRNAormiRRNAiintooneoftheBLOCK-iT™entry(shRNA)orexpression(miR

RNAi)vectors.

2. TransfertheRNAicassetteintotheadenoviral(shRNAonly)orlentiviraldestinationvectorbyGateway®recombination.

3. Transfectvectorsintotheappropriatekit-providedpackagingcells(useViraPower™PackagingMixforlentiviralsystemsonly)toproduce

viralstocks,whichcanbeusedimmediatelyorstoredat–80°C.

4. Harvestand(foradenovirusonly)amplifytheviralsupernatantanduseitforshRNA/miRRNAideliverytoanycelltype.

Store up to 1 year



Generate the pLenti expression construct containing the miR RNAi

or shRNA of interest


Packaging Mix




pLenti/BLOCK-iT™

RNAiconstruct

Ampicillin SV40 pA

RRE

pUC ori

PSV40

PRSV

/5´ L

TR

∆U3/

3´ L

TR

ψ

EM7

Selection

marker

Figure 8.12. How the BLOCK-iT™ lentiviral system works.

Table 8.4. Choose a lentiviral or adenoviral RNAi system.

Viral system When to use Products

LentiviralRNAideliverysystems

• StableRNAiinanycellline,evennon-dividingcells

• InducibleorconstitutiveshRNAormiRRNAiexpression

• Studiesinanimalmodels

• BLOCK-iT™LentiviralPolIImiRRNAiExpressionSystem—acompletelentiviralsystemwithalloftheadvantagesofmiRRNAi:multiple-targetknockdownandahigherdesignsuccessratethanconventionalshRNA(containspLenti6/V5-DEST™vector)

• BLOCK-iT™LentiviralPolIImiRRNAiExpressionSystemwithEmGFP—asystemwithallofthebenefitslistedabove,pluseasyexpressiontrackingwithcocistronicEmGFP(containspLenti6/V5-DEST™vector)

• BLOCK-iT™InducibleH1LentiviralRNAiSystem—completelentiviralsystemforinducibleorconstitutiveshRNAexpressioninanycelltype(containspLenti4/BLOCK-iT™-DESTvector)

• BLOCK-iT™LentiviralRNAiExpressionSystem—completelentiviralsystemforconstitutiveshRNAexpressioninanycelltype(containspLenti6/BLOCK-iT™-DESTvector)

BLOCK-iT™RNAiAdenoviralSystem

• High-leveltransientshRNAexpression• Effectivedeliverytoawiderangeof

humancelltypes• Studiesinanimalmodels

• BLOCK-iT™AdenoviralRNAiExpressionSystem—completesystemforhigh-leveltransientexpressionofshRNA

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Powerful shRNA delivery with BLOCK-iT™ viral vectors

Theviraldeliverysystemsincludethesevectors,whicharealsoavailableseparately(Figure8.13):

→ pLenti4/BLOCK-iT™-DEST—lentiviral vector with the Zeocin™ selection marker for compatibility with a tetracycline-regulated inducible

system

→ pLenti6/BLOCK-iT™-DEST—lentiviralvectorwiththeblasticidinselectionmarkerforfast,efficientselectionforconstitutiveshRNAexpression

→ pAd/BLOCK-iT™-DEST—adenoviral vector expediently produced without the need for a shuttle vector or for performing other labor-

intensivemethods

Product Quantity Cat. No.BLOCK-iT™LentiviralPolIImiRRNAiExpressionSystem 20rxns K4937-00

BLOCK-iT™LentiviralPolIImiRRNAiZ´-factorExpressionSystemwithEmGFP 20rxns K4938-00

BLOCK-iT™InducibleH1LentiviralRNAiSystem 20rxns K4925-00

BLOCK-iT™LentiviralRNAiExpressionSystem 20rxns K4944-00

BLOCK-iT™AdenoviralRNAiExpressionSystem 20rxns K4941-00


8.0 kb

attR1 attR2ccdB CmR


8.7 kb


3.5 kb

attR1 attR2CmR ccdBattR1 attR2CmR ccdB

PR

SV/5

´ LTR

RRE

wt A

d5 (∆

E3)

Am

pici

llin5´ ITR

pU

C ori

3´ ITR3´ LTR (∆

E3)

Bla

stic

idin

SV40 pA

Am

picillin

pU

C ori

PSV40

EM7

3´ LTR (∆E3

)Z

eoci

n™

SV40 pA

Am

picillin

pU

C ori

PR

SV/5

´ LTR

RRE PSV40

EM7

Figure 8.13. BLOCK-iT™-DEST viral vectors. TheBLOCK-iT™InducibleH1LentiviralRNAiSystemcangeneratelong-terminducibleknockdownresultswhetheraclonalpopulationisusedorindividualclonesareselectedforanalysis.ThepromoterconfigurationoftheH1/TOvectorensurestightregulation;thus,unwantedbackgroundfromshRNAexpressionwillnotinterferewithresults.




BLOCK-iT™ Lentiviral RNAi System

Stable RNAi expression in nondividing mammalian cellsTheRNAicassette fromtheBLOCK-iT™U6entryvectorcanalsobe transferred into thepLenti6/BLOCK-iT™-DESTorpLenti4/BLOCK-iT™-DEST

lentiviralRNAivectors.First,generatelentiviralstockswiththe293FTcelllineandassociatedViraPower™reagents,whichareavailableseparately

orwiththeBLOCK-iT™LentiviralRNAiSystem.Then,preparealentiviralstock,transducecells,andassayfortheRNAiresponse(Figures8.14and

8.15).TheBLOCK-iT™lentiviralRNAivectorspossessalltherequiredcomponentsforefficientpackagingoftheRNAishRNAtodividing,hard-to-

transfect,andnondividingcells,aswellasforin vivo deliverytoanimalmodels.

Mag

icM

ark™

Mag

icM

ark™

Mag

icM

ark™

Mag

icM

ark™

Day 6Day 3 Day 60Day 23 Day 37Day 16

– + – + – + – + – + – + – + – + – + – +– + – +lamAC lacZlamAC lacZ lamAC lacZ lamAC lacZ lamAC lacZ lamAC lacZshRNA:

1 µg/ml Tet:

Lamin A/C

β-actin

120100

80

60

50

40

30

120100

80

60

50

40

30

120100

80

60

50

40

30

120100

80

60

50

40

30

Figure 8.15. Long-term inducible knockdown in stably transduced T-REx™ HeLa cells. T-REx™HeLacellsweretransducedwithlentiviralparticlesexpressinglaminA/C(lamAC)-directedorcontrollacZ-directedshRNAfromthetetracycline(Tet)-inducibleH1/TOpromoterataneffectiveMOIof<1.StablepopulationsofcellswereselectedwithZeocin™reagentfor3weeksandplatedwithorwithoutTetat1μg/mLforthenumberofdaysindicated.WholecelllysateswereseparatedbywesternblottingandprobedwithantibodiesagainstlaminA/Candβ-actinasaloadingcontrol.

Store up to 1 year



Generate the pLenti expression construct containing the

gene of interest


Packaging Mix




Pol III promoter Termination sequenceshRNA sequence

pLenti/BLOCK-iT™

RNAiconstruct

Ampicillin SV40 pA

RRE

pUC ori

PSV40

PRSV

/5´ L

TR

∆U3/

3´ L

TR

ψ

EM7

Selection

marker

Figure 8.14. Generating BLOCK-iT™ lentiviral RNAi stock.

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34.9 Kb

Generate the adenoviralRNAi vector containing the

shRNA of interest. Digest thepuri�ed plasmid with Pac I to expose the inverted terminal repeats (ITRs).

Transfect the 293Aproducer cell line with

the adenoviral RNAi vector.Harvest cells and prepare

a crude viral lysate.

Amplify the adenovirusby infecting 293A producer

cells with the crude virallysate. Determine the titerof the adenoviral stock.

Add the viral supernatantto the mammalian cell

line of interest.

Assay for knockdown of gene on interest

293A cell line Target mammalian cell line

Promoter shRNA sequence

Pac I

Pac I

Pol III promoter Termination sequenceshRNA sequence

pUC

ori

3´ ITR

Am

pici

llin

5´ ITR + 3´ ITR

wt Ads ( ∆E3)

Figure 8.16. How the BLOCK-iT™ adenoviral RNAi system works.

Product Quantity Cat. No. BLOCK-iT™InducibleH1RNAiEntryVectorKit 20constructions K4920-00

BLOCK-iT™U6RNAiEntryVectorKit 20constructions K4945-00

T-REx™-JurkatCellLine 3x106cells R722-07

T-REx™-CHOCellLine 3x106cells R718-07

T-REx™-HeLaCellLine 3x106cells R714-07

T-REx™-293CellLine 3x106cells R710-07

pLenti6/TRVectorKit 1kit V480-20

pcDNA™6/TR 20µg V1025-20

BLOCK-iT™AdenoviralRNAiExpressionSystem 20rxns K4941-00

pAd/BLOCK-iT™-DESTRNAiGateway®Vector 6µg V492-20

293ACellLine 3x106cells R705-07

BLOCK-iT™ Adenoviral RNAi Expression System

Efficient transient delivery and shRNA expression in mammalian cellsAdenoviralsystemsarepopularplatformsforreliablegenedeliveryandsuperiorexpressionoftransientshRNAinnearlyanymammaliancelltype.

ThekeyadvantageoftheBLOCK-iT™AdenoviralRNAiExpressionSystemisGateway®recombinationtechnology,whichsimplifiesthecloning

andgenerationofanadenoviralvector,eliminatingthetediousandtime-consumingmanipulations,screening,andmultipletransformationsthat

otheradenoviralsystemsrequire.

Adenovirusesenter targetcellsbybindingtotheCoxsackieadenovirus receptor (CAR) [8].AfterbindingtoCAR, theadenovirus is inter-

nalizedvia integrin-mediatedendocytosis [9], followedbyactivetransport tothenucleuswheretheshRNA isexpressed.High-level transient

shRNAexpressioniseasilyachievedwiththeBLOCK-iT™AdenoviralRNAiSystem(Figure8.16).Gateway®recombinationprovidesawaytoquickly

generateanadenoviralRNAiconstruct.Forthefirsttime,high-throughputcloningintoadenoviralvectorsispossibleduetotheefficacyofthis

recombinationreaction.

1. BrummelkampTRetal.(2002)Science 296:550–553.

2. McManusMTetal.(2002)RNA 8:842–850.

3. PaddisonPJetal.(2002)Genes Dev 16:948–958.

4. SuiGetal.(2002)Proc Natl Acad Sci U S A 99:5515–5520.

5. YuJYetal.(2002)Proc Natl Acad Sci U S A 99:6047–6052.

6. GwizdekCetal.(2003)J Biol Chem 278:5505–5508.

7. YiRetal.(2003)Genes Dev 17:3011–3016.

8. BergelsonJMetal.(1997)Science 28:1320–1323.

9. RussellWC(2000)J Gen Virol 81:2573–2604.

Chapter references




CHAPTER 9

RNAi delivery

Introduction to RNAi delivery

ThetwomostcommonapproachesforRNAideliveryare lipid-mediatedtransfectionandvirus-mediatedtransduction.Determiningwhichof

theseapproachestousedependsonthecell typebeingstudiedandwhethertransientorstableknockdownisdesired(Table9.1).Themost

popularapplication,transienttransfectionofunmodifiedsiRNAs,modifiedsiRNAduplexes,orRNAivectors,utilizescationiclipid–basedreagents

becausetheyaresuitablefordeliveringthesemoleculesacrossadiverserangeofcommonlyusedcelllines.Forcelltypesnotamenabletolipid-

mediatedtransfection,viralvectorsareoftenemployed.Adenoviralvectorsworkwellfortransientdeliveryinmanycelltypes;however,forstable

deliveryindividingandnondividingcells,lentiviralvectorsarebest.TodeterminethemostfavorableRNAideliveryconditions,Invitrogenoffers

youourdeliveryoptimizationservice (seeChapter12),a resourcebackedbyextensiveknowledgeandexpertise inviralvectorsandnonviral

deliveryreagentsfortestingamatrixofdeliveryparameters.

Methods to achieve high transfection efficiencyTransfectionefficiencyisexpressedasthepercentageofcellsthathavereceivedtheRNAiduplexorexpressionplasmid.Typically,researchers

strive toachieve thehighestefficienciespossible. Thisobjective isparticularly important forRNAiapplications,becausenontransfectedcells

willcontinuetoexpressnormallevelsofthetargetedgene,whichwillresultinbackgroundexpressionlevels.WiththecationicLipofectamine®

RNAiMAXTransfectionReagent,transfectionefficiencycanbeoptimizedandverifiedeasilyusingtheBLOCK-iT™AlexaFluor®RedFluorescent

Control.InthecaseofLipofectamine®2000TransfectionReagent,eithertheBLOCK-iT™AlexaFluor®RedFluorescentControlortheoriginalgreen

BLOCK-iT™FluorescentOligocanbeused.

Forsomediseasemodels,primaryculturesornondividingcells,whichareoftenthemostappropriatecelltypestouse,cannotbetrans-

fectedadequatelywithcommerciallyavailabletransfectionreagents.Apowerfulalternativetocationiclipid–mediatedtransfectionisviraldeliv-

eryofvectorscontainingRNAicassettes.Thisoptionisbestfordeliverytohard-to-transfect,primary,andnondividingcells.Viraldeliverycan

alsobeusedtocreatestablecelllineswithinducibleRNAiortoexpressRNAisequenceswithtissue-specificpromoters.SeeChapter4formore

informationaboutusingviralvectorsinRNAiexperiments.

Importance of minimizing transfection-mediated cytotoxicityThedeliveryofsiRNAs,or themethodofdelivery,can result incytotoxicity ingenesilencingexperiments.Minimizingtransfection-mediated

cytotoxicityisessentialforproperinterpretationoftheoutcomeofanyRNAiexperiment.Cytotoxiceffectscanbedifficulttodistinguishfrom

theexpectedphenotyperesultingfromtargetgeneknockdown.Cytotoxicityduetothedeliverymethodisoftenseenasapparentknockdown

Table 9.1. Selecting transfection reagents and viral delivery methods.

Product Key advantages

Lipofectamine®RNAiMAXTransfectionReagent

• DesignedandmanufacturedforsiRNAdelivery• Superiorefficiencies,allowinglowconcentrationsofsiRNAtobeused• Widerangeofcompatibilitywithdiversecelllines• Optimizedprotocolsavailableformanycommoncelllines

Lipofectamine®2000TransfectionReagent• Designedforoptimalexpressionwhendeliveringplasmids,includingshRNAandmiRRNAivectors• Robustcotransfectionofvectorsandsynthetics(siRNAorStealthRNAi™siRNAduplexes)

Lipofectamine®LTXReagent• Designedforimprovedtransfectionperformanceinhard-to-transfectandprimarycells• Hightransfectionefficiencyandsignificantlylowertoxicitylevelsforawiderangeofcelllines

Oligofectamine™TransfectionReagent• Expresslyformulatedfordeliveryofantisenseoligos• DependabledeliveryofsiRNA

BLOCK-iT™AdenoviralRNAiExpressionSystem • Idealsystemforlong-termtransientexpressionofRNAivectorsinawiderangeofcelllines

BLOCK-iT™LentiviralPolIImiRRNAiExpressionSystemBLOCK-iT™LentiviralPolIImiRRNAiExpressionSystem withEmGFPBLOCK-iT™LentiviralRNAiExpressionSystemBLOCK-iT™InducibleH1LentiviralRNAiSystem

• StableexpressionofRNAivectorsindifficult-to-transfectcelllines• Suitablemethodsforin vivoapplications• Induciblesystemsavailable

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Chapter 9RNAi delivery

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incellstransfectedwithseveralnegativecontrols,relativetonotreatment.Theeasiestwaytocombattransfection-mediatedcytotoxicityisto

chooseatransfectionreagentdesignedforeitherdouble-strandedRNAorplasmid-basedRNAitransfections.Mostoften,thesereagentshave

beenformulatedtomaximizeknockdownefficiencywhileminimizingcytotoxicity.Optimizationexperiments,whenusingpublishedorsuppli-

ers’protocolsasguidelines,canhelptodeterminetheconcentrationoftransfectionreagentthatworksbestforthecelllineofinterest.Usingthe

lowestamountoftransfectionreagentneededtogivethehighestlevelofknockdownisrecommended.

Significance of reducing off-target effectsAswellasbeingasourceofcytotoxicity,asuboptimaltransfectionreagentorexcessreagentcanresultinapparentoff-targeteffects.Onecause

ofoff-targeteffectsistheup-ordown-regulationofgenesduetothegenedeliveryprocedure.However,withappropriatecontrols,theseeffects

canbe identifiedanddiminished (seeChapter2 foradiscussionofcontrols forRNAiexperiments).Again, the lowestamountof transfection

reagentthatprovidesthebestgenesilencingactivityisadvised.

Thepotentialalsoexistsforoff-targeteffectscausedbythesiRNAduplexitself.Todeterminethemostfavorableconditionsfortransfection,

theconcentrationofsiRNAshouldbevariedwhileholdingtheconcentrationoftransfectionreagentconstant,usingthelowestconcentration

(identifiedpreviously)tolimitcytotoxicity.KeepinmindthatthespecificityofthesiRNAwillimpactpotentialoff-targeteffects.Theuseofexcep-

tionallyspecificreagents,suchasmodifiedStealthRNAi™siRNAduplexes,canhelpalleviatetheseconcerns(seeChapter7fordetailedinforma-

tionaboutthespecificityadvantageofStealthRNAi™siRNA).

Lipofectamine® RNAiMAX Transfection Reagent—unmatched gene silencing with reduced cytotoxicity for siRNA experimentsLipofectamine®RNAiMAXTransfectionReagentisaproprietary,siRNA-specificcationiclipidformulationthatoffersthehighesttransfectioneffi-

ciencieswiththewidestvarietyofcelltypesforsiRNAgeneknockdownexperiments:

→ SuperiortransfectionefficiencyenablesuseoflowersiRNAconcentrationsandleadstomoresuccessfulgeneknockdownwithaminimum

ofnonspecificeffects(Figure9.1A)

→ Easyoptimizationduetolowcytotoxicityacrossa10-foldrangeoftransfectionreagentconcentrations(Figure9.1B)

→ Versatileapproachcompatiblewithawiderangeofcelltypes

→ Simpleandrapidprotocolforconsistentresults

Stealth RNAi™ siRNA concentration

0.5 nM50 nM 1 nM10 nM1 nM0.5 nM 10 nM 50 nM Cells only

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2.0

1.5

1.0

0.5

0

Stealth RNAi™ siRNA Negative Control Medium GC

Human p53 Stealth RNAi™ siRNA

p53 knockdown % viable cells

Lipofectamine® RNAiMAX reagent volume

00.1 µL0.25 µL0.5 µL1.0 µL 00.1 µL0.25 µL0.5 µL1.0 µL

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53 v

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Per

cent

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1.4

1.0

0.6

0.2

1.2

0.8

04

0

120

80

60

20

100

40

0

1 nM Stealth RNAi™ siRNA NegativeControl Medium GC

1 nM p53 Stealth RNAi™ siRNA

Figure 9.1. Optimal gene silencing and minimal cytotoxicity with Lipofectamine® RNAiMAX Transfection Reagent. (A)Superiorknockdownlevelswereobservedwithaslittleas0.5nMStealthRNAi™siRNAwithLipofectamine®RNAiMAXTransfectionReagent.Transfectioncomplexescontaining0.3μLofLipofectamine®RNAiMAXTransfectionReagentwerepreparedin48-wellplates.A549cellswereaddedtoeachwelltogivefinalStealthRNAi™siRNAconcentrationsof0.5to50nM.Twenty-fourhoursafteradditionofcells,p53knockdownwasmeasuredbyqRT-PCRwithLUX™primersandnormalizedtoGAPDHexpres-sion.ThecontrolduplexwastheStealthRNAi™NegativeControlMediumGC.(B)Minimalcytotoxicityovera10-foldrangeofLipofectamine®RNAiMAXTransfectionReagentconcentrations. Indicatedvolumesof transfection reagentweremixedwith10nMp53ValidatedStealthRNAi™siRNAorStealthRNAi™NegativeControlMediumGCduplexesin48-wellplates.A549cellswereaddedtoeachwellforafinalsiRNAconcentrationof1nM.Knockdownofp53wasmeasuredasdescribedin(A).Overa10-foldconcentrationrangeofLipofectamine®RNAiMAXTransfectionReagent,highlevelsofgenesilencingwereobtainedwithoutadramaticincreaseintransfection-mediatedcytotoxicity.

A B

9RNAi delivery



Product Quantity Cat. No.Lipofectamine®RNAiMAXTransfectionReagent 0.75mL

1.5mL13778-07513778-150

BLOCK-iT™AlexaFluor®RedFluorescentControl 2x125μL 14750-100

RNA interferenceSimple protocol. TheprocedureforusingLipofectamine®RNAiMAXTransfectionReagentconsistsofmixingitwithsiRNA,addingcells,incubat-

ing,andmeasuringgeneknockdown(Figure9.2).Thesimplicityandspeed,combinedwithsuperiortransfectionefficiency(Figure9.3),make

Lipofectamine®RNAiMAXTransfectionReagentthebestchoiceforhigh-throughputsiRNAtransfections.Transfectionconditionscanbereadily

establishedforautomatedorroboticsystemsusedinsuchapplications.ProtocolsforusingtheLipofectamine®RNAiMAXTransfectionReagent

canbefoundatwww.invitrogen.com/rnaimax.

Figure 9.3. Assessing transfection efficiency of the Lipofectamine® RNAiMAX Transfection Reagent with the BLOCK-iT™ Alexa Fluor® Red Fluorescent Control. Lipofectamine®RNAiMAXTransfectionReagentwasusedtotransfectHeLacellswiththeBLOCK-iT™AlexaFluor®RedFluorescentControl (50nM) (A).Twenty-fourhoursaftertransfection,growthmediumwasremovedandreplacedwithPBScontaining10mg/mLHoechst33342forvisualizationofcellnuclei(B).Nuclearlocalizationofthered-fluorescentcontrololigoisseen(A).Thebright-fieldimage(C)showsthatthecellsretainanormalmorphologyaftertransfection.

A B C

HASMC NTERA-2 HUVECHREHEKa ADSC Control

100

20

15

10

5

0

GAPDH

CSNK2A1

Rel

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Figure 9.2. Superior knockdown of Silencer® Select siRNA with Lipofectamine® RNAiMAX Transfection Reagent. ([email protected]).

NOTE: Lipofectamine® RNAiMAX Transfection Reagent is for use with siRNA duplexes, and not for transfecting vectors expressing RNAi sequences.

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NOTE : The BLOCK-iT™ Fluorescent Oligo (Cat. No. 2013) is optimized for use with Lipofectamine® 2000 Transfection Reagent and does not work well with Lipofectamine® RNAiMAX Transfection Reagent.

Figure 9.4. Delivery of BLOCK-iT™ Pol II miR RNAi expression vectors. Cells were transfected with pcDNA™6.2-GW/EmGFP-miR (directed against lamin) andLipofectamine®2000TransfectionReagent,atanexpected50%efficiency,todemonstratethe100%trackingofEmeraldGreenFluorescentProtein(EmGFP)andmiRNAexpression.After48hr,cellswerestainedwithHoechstnuclearstain,whichstainsallcells(A),andaredlaminstain(B),andmonitoredforGFPexpression.Approximatelyhalfofthecellshighlyexpressedthelaminprotein,butred-stainedcellswerenotexpressingEmGFP,andEmGFP-expressingcellswerenotexpressinglamin(C).ThisdemonstratesthecocistronicexpressionofEmGFPandthemiRNAthatgreatlyreduceslaminexpression.

Product Quantity Cat. No.Lipofectamine®LTXReagent 1mL 15338100

PLUS™Reagent 0.85mL 11514015

Lipofectamine®2000TransfectionReagent 0.75mL1.5mL

11668-02711668-019

BLOCK-iT™FluorescentOligoforlipidtransfection 2x125μL 2013

BLOCK-iT™TransfectionKit* 1kit 13750070

*TheBLOCK-iT™TransfectionKitcontainsLipofectamine®2000TransfectionReagentandtheBLOCK-iT™FluorescentOligo.

A. Hoechst B. Lamin C. EmGFP + lamin

Lipofectamine® 2000 and LTX Transfection Reagents—ideal for delivery of shRNA and miR RNAi vectorsLipofectamine®2000TransfectionReagentcanbeusedfordeliveryofassortedRNAiagents,includingshRNAandmiRRNAivectorsandsynthetic

moleculessuchassiRNA,StealthRNAi™siRNA,andDicer-generatedsiRNApools.Lipofectamine®LTXReagent,usedincombinationwithPLUS™

Reagent, is thechoicereagentforhard-to-transfectcell lines, includingprimarycells, fordeliveryofassortedRNAimolecules.Lipofectamine®

2000andLipofectamine®LTXreagentsforRNAitransfectionoffermanybenefits:

→ Effective transfection for shRNA and miR RNAi vectors (Figure 9.4) and synthetics (siRNA and Stealth RNAi™ siRNA); also works well for

cotransfectionsofsyntheticsandvectors

→ Easy-to-followprotocols;mediachangesnotrequired

→ ConvenientoptimizationoftransfectionconditionsandefficiencywiththeBLOCK-iT™FluorescentOligo

→ Excellentperformanceinawidevarietyofcelltypes

9RNAi delivery



Oligofectamine™ Transfection Reagent—potent internalization of RNA oligos

Oligofectamine™TransfectionReagentisdesignedtodeliverantisenseoligosandisalsousefulfortransfectingRNAireagentsintoeukaryoticcells.

Tooptimizetransfectionconditions,Oligofectamine™TransfectionReagentcanbeusedinconjunctionwiththeBLOCK-iT™FluorescentOligoor

theBLOCK-iT™RNAiBasicControlKit.InthefirstpublisheddemonstrationofsiRNA-mediatedknockdowninamammaliancellline,Elbashiretal.

reportedtheiruseofOligofectamine™TransfectionReagenttotransfectHeLacellswithsiRNAs[1].ThisworkwascontinuedbyHarborthetal.,

whousedasimilartransfectionprotocolwithOligofectamine™TransfectionReagenttodemonstratetheknockdownof20additionalessential

andnonessentialtargetgenes[2].TheseearlystudiesestablishedthegeneralapplicabilityofusingsiRNAforRNAi.

Product Quantity Cat. No.Oligofectamine™TransfectionReagent 1.0mL 12252-011

The Neon™ Transfection System

High transfection efficiency and high cell viability in a broad range of cell lines

Thecompanythatcontinuestobringyoutransfectionreagentsonthecuttingedgenow

introducestheNeon™TransfectionSystem.

→ Efficiency—up to 90% in many cell types, including difficult-to-transfect, primary,

andstemcells(Table9.2)

→ Flexibility—easilytransfectfrom2x104cellsto6x106cellsperreaction

→ Simplicity—singlereagentkitforallcelltypes

→ Versatility—opensystemallowselectroporationparameterstobeoptimizedfreely

TheNeon™TransfectionSystemisanovelbenchtopelectroporationdevicethatemploysanelectroporationtechnologyusingapipettetipas

anelectroporationchambertoefficientlytransfectmammaliancells, includingprimaryand immortalizedhematopoieticcells,stemcells,and

primarycells(Figure9.5).TheNeon™TransfectionSystemefficientlydeliversnucleicacids,proteins,andsiRNAintoallmammaliancelltypeswith

ahighcellsurvivalrate.Thetransfectionisperformedusingasfewas2x104orasmanyas8x106cellsperreaction,inasamplevolumeof10μL

or100μL,inavarietyofcellcultureformats(60mm,6-well,48-well,and24-well).

TheNeon™TransfectionSystemusesasingletransfectionkit(Neon™Kit)thatiscompatiblewithvariousmammaliancelltypes,including

primaryandstemcells,therebyavoidingtheneedtodetermineanoptimalbufferforeachcelltype.

TheNeon™TransfectionSystemoffersopenandtransparentprotocolsthatareoptimizedforeaseofuseandsimplicity.TheNeon™device

ispreprogrammedwithone24-welloptimizationprotocoltooptimizeconditionsforyournucleicacid/siRNAandcelltype,oryoucanprogram

andstoreupto50cell-specificprotocolsintheNeon™devicedatabase.Optimizedprotocolsformanycommonlyusedcelltypesarealsoavailable

atwww.invitrogen.com/neonforyourconvenience,tomaximizetransfectionefficienciesforyourcelltypes.

Neon™ system pipette tip design vs. standard electroporation cuvetteUnlikestandardcuvette-basedelectroporationchambers,theNeon™systemusesapatentedbiologicallycompatiblepipettetipchamberthat

generatesamoreuniformelectricfield(Figure9.6).Thisdesignallowsbettermaintenanceofphysiologicalconditions,resultinginveryhighcell

survivalcomparedtoconventionalelectroporation[3].

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Figure 9.6. Standard electroporation cuvette compared to Neon™ system pipette tip. Thedesignoftheelectrode in thepipettehasbeenshown toproduceamoreuniformelectricfield. Theresultislesstoxicitytothecellsandhighertransfectionefficiencies.

Table 9.2. Examples of cell lines successfully transfected using the Neon™ Transfection System.

Cell line Cell type Transfection efficiency (%)* Viable cells (%)

MEFprimary Embryonicfibroblast 80 75

293A Kidney 90 90

3T3-L1 Mouseadipose 85 80

A549 Lung 75 92

Macrophages Human(peritoneal) 60 60

MCF-7 Breast 70 80

HeLa Cervicalcarcinoma 90 87

HL-60 Blood 55 70

PBMC Blood 23 95

Primaryratcorticalcells Brain,cortical 42 98.5

Primaryrathippocampalcells Brain,hippocampal 37 77

Raw264.7 Blood 74 80

* Transfectionefficiencyiscalculatedfromtotalpopulationofliveanddeadcells.ProtocolsandreferenceinformationforalargenumberofcelllinesareavailableintheNeon™celldatabaseatwww.invitrogen.com/neon.

Figure 9.5. High transfection efficiency of Jurkat cells with the Neon™ Transfection System. IntracelluaruptakeofreportervectorencodedwithEGFPat24hrfol-lowingtransfectionofJurkatcellsusingtheNeon™TransfectionSystem.BisthecorrespondingfluorescenceimageofA.

A B

9RNAi delivery



Chapter references1. ElbashirSMetal.(2001)Nature411:494–498.

2. HarborthJetal.(2001)J Cell Sci 114:4557–4565.

3. KimJA,ChoK,ShinMSetal.(2008)Biosens Bioelectron23(9):1353–1360.

1 2 3

Figure 9.7. Simple 3-step transfection procedure. 1.Loadmixofharvestedcellsandmoleculestobedelivered(e.g.,DNA,RNA,protein)intotheNeon™systempipettetip.2.PlugthepipetteintopositionintheNeon™transfectiondevice,selectyourprotocol,andpressStart.3.Unplugthepipetteandtransferyourtransfectedcellsintoatissueculturevessel.

Plug-and-play out of the boxThe transfectionprocessescouldn’tbesimpler (Figure9.7).TheNeon™systemusesa simple3-step transfectionprocedure: takeup thecells

andplasmidmixintotheNeon™systempipettetip,plugitintothepipetteholder,andpressStart.ThetransfectionoccursintheNeon™system

pipettetip.Then justpipette thetransfectedcells intoyourculturevessel.Nomorefillingandpullingsample fromthecuvetteandcapping

anduncapping.

Simplified transfection kit for all cell typesAvoidthehassleofdeterminingwhichproprietarybufferkitwillworkwithyourfavoritecelltype.Wehavesimplifiedyourworkwithjustone

transfectionkitthat iscompatiblewithallcelltypes.Startyourtransfectionwithouroptimizedprotocolsforpopularcelltypes,orfollowour

standardsimpleoptimizationprocedureforallnewcelltypes.

Product Quantity Cat. No.Neon™TransfectionSystem100µLKit 192rxns MPK10096

Neon™TransfectionSystem10µLKit 192rxns MPK1096

Neon™TransfectionSystem 1each MPK5000

Neon™TransfectionSystemStarterPack 1pack MPK5000S

Neon™TransfectionSystemPipette 1each MPP100

Neon™TransfectionSystemPipetteStation 1each MPS100

Neon™TransfectionSystemExtendedWarranty 1each MPSERV

Neon™TransfectionTubes 1pack MPT100

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Chapter 10RNA interference controls

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CHAPTER 10

RNA interference controls


Overview of controls for RNAi experimentsAppropriatecontrolsareessential tosuccess ineveryRNAiexperiment.Thenumberandtypesofcontrolschosendependupontheultimate

researchgoal[1].WithInvitrogen’sRNAitechnologies,performingtheappropriatecontrolreactionshasbeensimplified(Table10.1).RNAicontrol

kitsaredesignedtoassistresearchersinidentifyingandvalidatingdrugtargets,generatingpublishabledata,andaccomplishingthefollowing:

→ DeterminingwhichRNAireagentsdeliverthebestknockdownresults

→ Achievinggreaterknockdownbyoptimizingtransfectionprotocols

→ Savingtimebyconfirmingcellhealthearlyinanexperiment

→ ProceedingconfidentlywithRNAiexperimentsbycomparingtargetedRNAireagentstoasetofreagentsoptimizedforinhibitionofpositive

controlgenesinhumancells

→ UsingnegativecontrolsthatmatchtheGCcontentofthetargetmolecule

Table 10.1. Control kits ensure successful gene inhibition experiments.

Products/kits

Components

Fluorescent oligo

Transfection reagent

Dead-cell stain

Nuclear stain

RNAi positive controls

RNAi negative controls

qRT-PCR primers

BLOCK-iT™AlexaFluor®RedFluorescentControl

•

BLOCK-iT™FluorescentOligo •

BLOCK-iT™TransfectionKit• •

Silencer®SelectPositiveControlsiRNA•

Silencer®SelectNegativeControlsiRNA•

StealthRNAi™siRNANegativeControl•

StealthRNAi™siRNAPositiveControl(actin,GAPDH,cyclophilinB)

•

StealthRNAi™siRNAReporterControl•

BLOCK-iT™TransfectionOptimizationKit(Human)

• • • •

BLOCK-iT™PolIImiRValidatedmiRNAControlVector

• •

Applications

TracktransfectionMonitorexperimentalvariation

Measuretheeffectofexperimentalknock-downvs.background

AssessknockdownOptimizetransfectionconditions

Assesscellviability

10RNA interference controls



BLOCK-iT™ Alexa Fluor® Red Fluorescent Control and green BLOCK-iT™ Fluorescent Oligo

Toachievesignificantlevelsofspecificgeneinhibition,theStealthRNAi™siRNAorsiRNAduplexesmustbetakenupbytargetcells.Factorssuch

aspoorcellhealthandhighpassagenumbercannegatively impact transfectionefficiency.ThegreenBLOCK-iT™FluorescentOligoand the

BLOCK-iT™AlexaFluor®RedFluorescentControlallowyoutoeasilyvisualizetransfectionresultsand,thus,providekeycontrolsforStealthRNAi™

siRNAorstandardsiRNAtransfectionexperiments.ThesestabilizedRNAduplexeshelpdeterminetransfectionefficiencybyproviding:

→ Strong,easilydetectablesignalthatindicatestransfectionefficiency(Figure10.1)

→ Clear,persistentsignalthatexceedstheintensityofotherlabeledRNAduplexes

→ Predominantnuclearlocalizationtoconfirmthatthecontrolhasbeeninternalized

→ Uniquesequencesthathavenohomologytoanyknowngene,avoidinginductionofnonspecificoroff-targeteffects

ThegreenBLOCK-iT™FluorescentOligoandtheBLOCK-iT™AlexaFluor®RedFluorescentControlstronglycorrelatewithtransfectionefficiency

withStealthRNAi™,Silencer®Select,andSilencer®siRNAs,aswellasAmbion®Pre-miR™miRNAmimicsandAmbion®Anti-miR™miRNAinhibitors.

Figure 10.1. BLOCK-iT™ Alexa Fluor® Red Fluorescent Control for clear visualization of transfection results. HeLacellsweretransfectedwiththeBLOCK-iT™AlexaFluor®RedFluorescentControlusingeitherLipofectamine®2000(A–C)orLipofectamine®RNAiMAXTransfectionReagent(D–F).TherecommendedHeLacelltransfectionprotocolwasusedforeachreagent,andthefinalcontrolconcentrationwas50nM.Twenty-fourhoursaftertransfection,growthmediumwasremovedandreplacedwithPBScontaining10µg/mLHoechst33342forvisualizationofcellnuclei(BandE).Nuclearlocalizationofthecontrolisseenwithbothtransfectionreagents(AandD).Almost100%ofthecellstakeupthecontrol,andcellsretainanormalmorphology,asseeninthebright-fieldimages(CandF).

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BLOCK-iT™ Transfection Kit—simple optimization of Lipofectamine® 2000 transfection reagent conditionsThe BLOCK-iT™ Transfection Kit enables you to shorten the process of optimizing RNAi transfection conditions and monitoring transfection

variationineveryexperiment.Tooptimizetransfectionparameters,theBLOCK-iT™TransfectionKitcontainsLipofectamine®2000Transfection

ReagentandthegreenBLOCK-iT™FluorescentOligo(Figure10.2).

NOTE: The BLOCK-iT™ Fluorescent Oligo is optimized for use with Lipofectamine® 2000 Transfection Reagent and is not recommended for use with Lipofectamine® RNAiMAX Transfection Reagent.

Product Quantity Cat. No.BLOCK-iT™AlexaFluor®RedFluorescentControl(lipidtransfection) 2x125µL 14750-100

BLOCK-iT™FluorescentOligoforlipidtransfection 2x125µL 2013

BLOCK-iT™FluorescentOligoforelectroporation 75µL(1mM) 13750-062

BLOCK-iT™TransfectionKit 1kit 13750-070

Lipofectamine®RNAiMAXTransfectionReagent 1.5mL0.75mL

13778-15013778-075

Lipofectamine®2000TransfectionReagent 1.5mL0.75mL

11668-01911668-027

A B

Figure 10.2. Strong fluorescence signal to optimize transfection. A549cellsweretransfectedwithgreenBLOCK-iT™FluorescentOligoorstandardfluorescentlylabeledRNA,usingLipofectamine®2000TransfectionReagent.Twenty-fourhourslater,thefluorescentsignalhadpersistedinthecellstransfectedwiththegreenBLOCK-IT™FluorescentOligo(A).Incontrast,thefluorescentsignalwaslargelyundetectableincellstransfectedwiththestandardlabeledsiRNA(B).




Product Quantity Cat. No.Silencer®SelectGAPDHPositiveControlsiRNA 5nmol 4390849

Silencer®SelectGAPDHPositiveControlsiRNA 40nmol 4390850

Silencer®SelectGAPDHPositiveControlsiRNA,In VivoReady 250nmol 4404024

Silencer®SelectNegativeControl#1siRNA 40nmol 4390844


Silencer®SelectNegativeControl#1siRNA,In VivoReady 250nmol 4404020



Silencer® Select positive and negative control siRNAs

→ ValidatedsiRNAcontrolsforoptimizingsiRNAexperiments

→ GAPDHPositiveControlfunctionallytestedinseveralcommoncelllines

→ NegativeControlsfunctionallyproventohaveminimaleffectsoncellproliferationandviability

→ IncludeSilencer®SelectsiRNAmodificationsforenhancedspecificity

→ Foruseinhuman,mouse,andratcells

Silencer® Select GAPDH Positive Control siRNAOurextensivelyvalidatedpositivecontrolsiRNAtohuman,mouse,andratGAPDHservesmultiplefunctions.First,itisanideal“test”siRNAfor

thosejustbeginningsiRNAexperiments,becauseitisvalidatedtoworkinmultiplecelllines.Inaddition,becauseittargetsGAPDHmRNA,which

iscommonlyusedasaninternalcontrol,itseffectsareeasytoassay,andthusitprovidesanexcellenttooltomonitorsiRNAtransfectionefficiency

byreal-timeRT-PCRaswellaswiththeAmbion®KDalert™GAPDHAssayKit.TheSilencer®SelectGAPDHsiRNAincludesthesamemodificationsfor

reducingoff-targeteffectsasfoundinotherSilencer®SelectsiRNAs.

Silencer® Select Negative Control siRNAsNegativecontrolsiRNAs—siRNAswithsequencesthatdonottargetanygeneproduct—areessentialfordeterminingtheeffectsofsiRNAdelivery

onthecellandforprovidingabaselinetocomparesiRNA-treatedsamples.TherearetwoextensivelytestedSilencer®SelectNegativeControl

siRNAsforthispurpose.ThesesiRNAsincludethesamemodificationsforreducingoff-targeteffectsfoundinotherSilencer®SelectsiRNAsand

havenosignificantsequencesimilaritytomouse,rat,orhumangenesequences.ThesenegativecontrolsiRNAshavebeentestedbymicroarray

analysisandshowntohaveminimaleffectsongeneexpression. Inaddition,multiparametriccell-basedassayshaveconfirmedtheyhaveno

significanteffectoncellproliferation,viability,ormorphologyinthecelllinestested.

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Stealth RNAi™ siRNA negative controls—ideal negative controls for every experiment

StealthRNAi™siRNAnegativecontrolsprovidethemeanstomeasuretheeffectofaStealthRNAi™siRNAduplextargetedtoaspecificgene,com-

paredtobackground.Thesecontrolshavethefollowingfeatures:

→ Three levelsofGCcontent(low,medium,high),withthreesequencesavailableformatchingGCcontenttothatofexperimentalStealth

RNAi™siRNAduplexes.MultiplesequencesineachGCrangegiveevenmorecertaintyofresults.

→ Nohomologytoanyknownvertebrategenes

→ Testedsequencesdonotinducestressresponse

TheStealthRNAi™siRNANegativeControlKitcontainsallthreecontrols(low,medium,andhighGCcontent,excludingsequences#2and#3),and

eachisalsoavailableseparately.

Product GC content

Suitable for use with Stealth RNAi™ siRNA duplexes with the following

GC content Quantity Cat. No.StealthRNAi™siRNANegativeControlKit 1kit 12935-100

StealthRNAi™siRNANegativeControlLoGC 36% 35–45% 250µL 12935-200

StealthRNAi™siRNANegativeControlLoGCDuplex#2 36% 35–45% 250µL 12935-110

StealthRNAi™siRNANegativeControlLoGCDuplex#3 36% 35–45% 250µL 12935-111

StealthRNAi™siRNANegativeControlMedGC 48% 45–55% 250µL 12935-300

StealthRNAi™siRNANegativeControlMedGCDuplex#2 48% 45–55% 250µL 12935-112

StealthRNAi™siRNANegativeControlMedGCDuplex#3 48% 45–55% 250µL 12935-113

StealthRNAi™siRNANegativeControlHiGC 68% 55–70% 250µL 12935-400

StealthRNAi™siRNANegativeControlHiGCDuplex#2 68% 55–70% 250µL 12935-114

StealthRNAi™siRNANegativeControlHiGCDuplex#3 68% 55–70% 250µL 12935-115




Stealth RNAi™ siRNA reporter controls

Stealth RNAi™ siRNA reporter controls are ideal for RNAi experiments to optimize your transfection conditions in any vertebrate cell line.

Thesecontrolsare:

→ Designedtoefficientlyknockdowntheirintendedtargets

→ Nothomologoustoanythinginthevertebratetranscriptome

Product Quantity Cat. No.StealthRNAi™siRNAGFPReporterControl 250µL 12935-145

StealthRNAi™siRNALuciferaseReporterControl 250µL 12935-146

StealthRNAi™siRNAlacZReporterControl 250µL 12935-147

StealthRNAi™siRNAβ-LactamaseReporterControl 250µL 12935-148

Stealth RNAi™ siRNA positive controls

StealthRNAi™siRNApositivehousekeepingcontrolduplexes(human)areidealcontrolsforassessingknockdownandoptimizingRNAiexperi-

ments.Thesecontrolsare:

→ Designedtoefficientlyknockdowntheirintendedtargets

→ Bench-testedandsuppliedinaready-to-useformat

Product Quantity Cat. No.StealthRNAi™siRNAGAPDHPositiveControl(human) 250µL 12935-140

StealthRNAi™siRNAActinPositiveControl(human) 250µL 12935-141

StealthRNAi™siRNACyclophilinBControl(human) 250µL 12935-142

BLOCK-iT™ Transfection Optimization Kit (Human)

TheBLOCK-iT™TransfectionOptimizationKitisdesignedtooptimizeRNAitransfectionwithLipofectamine®2000TransfectionReagent,through

theuseofcontrolsfortransfectionandcellviability.EachkitcontainsaBLOCK-iT™FluorescentOligo,adead-cellstain,apositivecontrolStealth

RNAi™siRNAduplexdirectedathumanp53,andascrambledStealthRNAi™siRNAduplexasanegativecontrol.Thesereagents,usedtogether,

providethemeanstooptimizetransfectionconditionswithLipofectamine®2000TransfectionReagent,andafunctionalpositivecontrolforevery

RNAiexperiment.

Product Quantity Cat. No.BLOCK-iT™TransfectionOptimizationKit(human) 1kit 13750047

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Optimize RNAi experiments with the BLOCK-iT™ Fluorescent Oligo

Savingtimeandreagentsbynotrequiringseparateassaystobeperformedindifferentwells,theBLOCK-iT™FluorescentOligoallowsyoutoobtaina

clearvisualappraisalofRNAitransfectionefficiency.Thedead-cellstain,nuclearstain,andBLOCK-iT™FluorescentOligoemitsignalsatdifferentwave-

lengths,permittingthemtobedetectedtogetherinasinglewell.Thenuclearstain(Hoechstdye)anddead-cellreagent(ethidiumhomodimer-1)allow

assessmentofcelldeathandquantitationofcellnumber,makingsample-to-sampleandexperiment-to-experimentcomparisonsmoreaccurate.The

simpleadditionoftwodyestoanextracellculturesampleshowsthephysiologicalstateofcells.TheBLOCK-iT™FluorescentOligofortransfectionopti-

mizationandmonitoring,usedinconjunctionwithtwocell-stainingdyes,helpsachievethebestRNAiresultspossible(Figure10.3).

Transfectionefficienciesofatleast70%canbeconfirmedbythisprocedure:

1. Transfect human cell line with target-specific Stealth RNAi™ siRNA or siRNA duplexes, positive and negative controls, and BLOCK-iT™

FluorescentOligo.

2. Incubateaportionofthecellswithdead-cellstainandnuclearstain.

3. VisualizetheBLOCK-iT™FluorescentOligoandstainedcellstoassesstransfectionefficiencyandcellhealth;ifmorethan70%ofthecellsare

transfected(green)andcelldeathislessthan5%to10%,proceedtostep4.

4. MeasureRNAi-mediatedinhibitionoftargetedgenesusingTaqMan®GeneExpressionAssays(seeChapter11)

Product Size Cat. No.BLOCK-iT™FluorescentOligoforLipidTransfection 2x125µL 2013

BLOCK-iT™FluorescentOligoforElectroporation 2x125µL 13750062

Positive controls for BLOCK-iT™ Pol II miR RNAi vectors

TheBLOCK-iT™PolIImiRValidatedmiRNAControlVectorsarevaluablepositivecontrolsforinitialoptimizationandvalidationofexperimentsthat

usetheBLOCK-iT™PolIImiRRNAivectors(seeChapter8formoreinformationonthesevectors).Threevalidatedpositivecontrolsareavailable,

andeachcontroltargetsadifferentcommonlyusedreporter:lacZ,luciferase,orlaminA/C.ThesecontrolsareclonedintotheBLOCK-iT™miRRNAi

vectorswithEmeraldGFP(EmGFP)andarereadytouseforthefollowingapplications:

→ Optimizingtransfectionconditionsandmonitoringexperimentalvariationforrobustresults

→ IdentifyingthemostpotentreagentforoptimalRNAiknockdown

→ ValidatingmiRRNAiexpressionincommonlyusedreportersystems

Figure 10.3. Early cell visualization to assess quality of RNAi experiments. HeLacellsweretransfectedwith100nMBLOCK-iT™FluorescentOligo (green)and2.5mg/mLLipofectamine®2000TransfectionReagent.Twenty-fourhoursposttransfection,cellswerestainedwithdead-cell stain (ethidiumhomodi-mer-1, red)ornuclearstain (Hoechst33342,blue).Cellswereevaluatedat40xmagnificationbybright-fieldmicroscopyorwithappropriatefilters.TheblueHoechstdyestainsthenucleiofallcells,whiletheredethidiumhomodimer-1stainsonlydeadordyingcells.TransfectionefficiencyisdeterminedbycomparingtheBLOCK-iT™FluorescentOligotransfectioncontrolandnuclear-stainedcells;cellviabilityisdeterminedbycomparingthedeadcellsandnuclear-stainedcells.Ifcellviabilityislow,furthertransfectionoptimizationmayberequiredtoreducecelldeath.

Bright-field Fluorescent labeled oligoDead-cell stain Nuclear stain




Silencer® negative control siRNAs

→ ThesenontargetingsiRNAshavelimitedsequencesimilaritytoknowngenes

→ Validatedforuseinhuman,mouse,andratcells

→ Functionallyproventohaveminimaleffectsoncellproliferationandviability

→ Availableindividuallyandasapanelof7negativecontrolsiRNAs

→ HPLC-purified,duplexed,andreadytouse

Negative controlsNegativecontrolsiRNAs—siRNAswithsequencesthatdonottargetanygeneproduct—areessentialfordeterminingtransfectionefficiencyand

tocontrolfortheeffectsofsiRNAdelivery.InsiRNAscreeningapplications,negativecontrolsiRNAsarealsocriticalforsettingthe“hit”threshold

thatdetermineswhetheransiRNAisconsideredtohaveapositive,negative,orneutraleffectinaparticularassay.

WedesignedandtestedsevenNegativeControlsiRNAsthathavenosignificantsequencesimilaritytomouse,rat,orhumangenesequences.They

haveallbeentestedextensivelyincell-basedscreenstoensuretheyhavenosignificanteffectoncellproliferation,viability,ormorphology.Negative

Controls#1and#2,ourmostpopularnegativecontrolsiRNAs,areavailablein40nmoland5x40nmolsizes,inadditiontothestandard5nmolsize.

In Vivo Ready Negative ControlOur“In VivoReady”Silencer®NegativeControl#1siRNAissubjectedtoanextralevelofpurificationandtestingrequiredfortheintroductionofsiRNAsintoanimals.

AfterHPLCpurificationandannealing,eachsiRNAisfurtherpurifiedutilizingaprocessthatremovesexcesssaltviaasemipermeablemembrane.Theresultis

highlypuresiRNAwithminimalsaltcontent,suitableforin vivoapplications.In VivoReadysiRNAsarethenfilter-sterilized,andtestedforthepresenceofendo-

toxin.Atconcentrationsof50µMinthepresenceofdeionizedwater,In VivoReadysiRNAscontain<0.6mMNa+,<2.0mMK+,and<0.1mMMg2+.

Silencer® siRNA Screening Control PanelTheSilencer®siRNAScreeningControlPanelincludesallsevennegativecontrolsiRNAsandisintendedforlaboratoriesperformingsiRNAscreens

thatemployhundredsorthousandsofsiRNAsperexperiment.Becauseoftheimportanceofsuchscreensandthefactthatsomenegativecon-

trolsiRNAsmayhaveunintendedeffectsinaparticularassay,mostresearcherscarefullytesttheirnegativecontrolsiRNAsbeforechoosing2–6

controlsiRNAsforinclusionintheirsiRNAlibraryscreen.TheSilencer®siRNAScreeningControlPanelsimplifiesthisprocess.Asanaddedbenefit,

apositivecontrolsiRNAtohuman,mouse,andratKIF11(Eg5)isincludedinthepanel.KnockdownofKIF11,whichencodesakinesinfamilymotor

protein,leadstomitoticarrest,soeffectivedeliveryofKIF11siRNAcanbeassessedvisually;itcanalsobemeasuredbyseveralcell-basedassays.

TheSilencer®siRNAScreeningControlPanelcontains1nmolofeachofthesevennegativecontrolsiRNAsplustheKIF11siRNAinadried

format.InterestedinlargeramountsofanyNegativeControlsiRNA?Contactusatcustom_services@ambion.comforaquote.

Product Quantity Cat. No.Silencer®NegativeControl#1siRNA 5x40nmol AM4636

Silencer®NegativeControl#1siRNA 40nmol AM4635

Silencer®NegativeControl#1siRNA 5nmol(50µM) AM4611

Silencer®NegativeControl#1siRNA,In VivoReady 250nmol 4404021

Silencer®NegativeControl#2siRNA 5x40nmol AM4638

Silencer®NegativeControl#2siRNA 40nmol AM4637



Silencer®NegativeControl#4siRNA 5nmol(50 µM) AM4641



Silencer®siRNAScreeningControlPanel(includesnegativecontrolsiRNAs#1–7,plusKIF11siRNA) 8x1nmol AM4640

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Silencer® positive control siRNAs

→ ValidatedsiRNAcontrolsforoptimizingsiRNAexperiments

→ Gene-specificcontrolsiRNAsprovidedwithscramblednegativecontrols

→ Functionallytestedinseveralcommoncelllines

→ HPLC-purified,duplexed,andreadytouse

Premade, gene-specific siRNA controlsSilencer®controlsiRNAs,whichtargetmRNAsfrequentlyusedasinternalcontrolsinapplicationsdesignedtomonitorgeneexpression,suchas

RT-PCR,northernblot,andRPA,areidealfordevelopingandoptimizingsiRNAexperiments.EachSilencer®controlsiRNAisvalidatedforusein

humancelllines,includingtheGAPDHsiRNA,whichhasalsobeenoptimizedformouseandrat.(ThecyclophilinsiRNAhasalsobeenvalidated

inmousecell lines.) Formeasuringsilencingat theprotein level, a selectionofantibodiesareavailable, includingantibodiesagainstβ-actin,

cyclophilin,GAPDH,andKuproteins.EachSilencer®controlsiRNAcontains5nmolofready-to-usechemicallysynthesizedsiRNA.Ascrambled

negativecontrolsiRNA(2nmol)isincludedwithmostofthesesiRNAs.ThepopularSilencer®GAPDHsiRNAsarealsoavailablein40nmoland5x

40nmolsizes.

In Vivo Ready Positive ControlOur“In VivoReady”Silencer®GAPDHPositiveControlsiRNA issubjectedtoanextra levelofpurificationandtestingrequired for the introduc-

tionofsiRNAsintoanimals.AfterHPLCpurificationandannealing,eachsiRNAisfurtherpurifiedutilizingaprocessthatremovesexcesssaltvia

asemipermeablemembrane.TheresultishighlypuresiRNAwithminimalsaltcontent,suitableforin vivoapplications.In VivoReadysiRNAsare

thenfilter-sterilizedandtestedforthepresenceofendotoxin.Atconcentrationsof50µMinthepresenceofdeionizedwater,In VivoReadysiRNAs

contain<0.6mMNa+,<2.0mMK+,and<0.1mMMg2+.

Product Quantity Cat. No.Silencer®c-MycsiRNA 5nmol(dried) AM4250

Silencer®FireflyLuciferase(GL2+GL3)siRNA 5nmol+2nmolNegControl(50 µM) AM4629

Silencer®GAPDHPositiveControlsiRNA,In VivoReady 250nmol 4404025

Silencer®GAPDHsiRNA(Human) 5nmol+2nmolNegControl(50 µM) AM4605

Silencer®GAPDHsiRNA(Human) 40nmol AM4633

Silencer®GAPDHsiRNA(Human) 5x40nmol AM4634

Silencer®GAPDHsiRNA(Human,Mouse,Rat) 5nmol+2nmolNegControl(50 µM) AM4624

Silencer®GAPDHsiRNA(Human,Mouse,Rat) 5x40nmol AM4632

Silencer®GAPDHsiRNA(Human,Mouse,Rat) 40nmol AM4631

Silencer®GFP(eGFP)siRNA 5nmol+2nmolNegControl(50 µM) AM4626

Silencer®KIF11(Eg5)siRNA(Human,Mouse,Rat) 5nmol+2nmolNegControl(50 µM) AM4639

Silencer®RenillaLuciferasesiRNA 5nmol+2nmolNegControl(50 µM) AM4630

Chapter references1. Editors(2003)WhitherRNAi?Nat Cell Biol5:489–490.




CHAPTER 11

Measuring knockdown

Functional validation following RNAi knockdown

ForRNAiresultstobeconsideredvalid, relevant levelsoftarget-specificknockdownmustbedemonstrated. Ideally,measurementshowinga

substantialdecreaseofboththecellularmRNAandthetargetedproteinwouldbedemonstrated.Knowingthelevelofknockdowniscriticalfor

interpretationoffunctionalandphenotypicRNAieffects.Typically,atleast70%knockdown,asmeasuredbyquantitativereversetranscription

PCR(qRT-PCR),isconsideredtheminimumknockdownrequiredforasuccessfulRNAiexperiment.

Aknockdownlevelequaltoorexceeding70%mightgiveanegativephenotypicresult(i.e.,nochangeinphenotyperelativetocontrol),but

thisoutcomedoesnotnecessarilymeanthatthegeneofinterestisnotfunctionallyrelevant.Somegenesmaynotshowaphenotypicresponse

evenatgreatlyreducedlevelsofmRNA.Thislackofanobservableresponsecanbeduetotheproteinhavingalonghalf-life(thus,measuring

knockdownbywesternblottingmightbedesirable)orduetoaverysmallamountofproteinbeingsufficienttoachievenormalcellularfunction.

Ifthelatteristhecase,itisimperativetogeneratethehighestpossiblelevelofknockdowntoseeaneffect.

TaqMan® Gene Expression Assays

→ Gene-specificTaqMan®probeandprimersetsforquantitativegeneexpressionstudiesinhuman,mouse,rat,and16otherspecies

→ Convenientsingle-tubeformatand20Xformulation

→ Universalthermalcyclingconditions

TaqMan®GeneExpressionAssays(Figure11.1)areacomprehensivecollectionofover1.2millionpre-designedprimerandTaqMan®probesets

designedtoquicklyandeasilyperformquantitativegeneexpressionstudiesonhuman,mouse,rat,and16otherspecies.Eachgeneexpression

assayconsistsofaFAM™dye–labeledTaqMan®MGBprobeandapairofPCRprimersdesignedtoamplifythetargetofinterestfromcDNA.Assays

aresuppliedinapreformulated20Xmixinready-to-usesingletubes.Everyassayhasbeenoptimizedtorununderuniversalthermalcyclingcon-

ditionsatafinalreactionconcentrationof250nMfortheprobeand900nMforeachprimer.Wehavecombinedacomprehensiveassayselection

withastreamlinedapproachtoprovideyouwithaconvenient,standardizedprocessforquantitativegeneexpression.

Approximately60,000TaqMan®GeneExpressionAssaysforhuman,mouse,rat,andsevenotherspeciesareavailableasinventoried,off-

the-shelfproducts.Thiscollectionincludesanaverageof1assayforeachRefSeqmRNAtranscript.Approximately1.1millionadditionalassays

areavailableonamade-to-orderbasis,andcovermostexon-exonjunctionsofmRNAtranscriptsforhuman,mouse,rat,C. elegans,Drosophila,

Arabidopsis, and13otherspecies.

OurextensiveonlinecatalogofTaqMan®GeneExpressionAssayscanbeaccessedatwww.appliedbiosystems.com.Youcansearchforyourassay

usingpublicIDnumbers(includingRefSeqID,EntrezGeneID,orUnigeneID),commongenenames,symbols,oraliases,andfunctionalcategoriesand

groups(suchaskinases,cytokines,transcriptionfactors,etc.).ForacompletelistofTaqMan®GeneExpressionAssaysinsingle-tubeformat,orpreloaded

in96-and384-wellplatesandmicrofluidiccards,visitwww.allgenes.com.

Figure 11.1. TaqMan® Gene Expression Assays are delivered with a compact disc containing an electronic assay information file.

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Custom TaqMan® Gene Expression Assays

→ Availableforanyspeciesororganism

→ Usethetargetsequenceofyourchoice

→ Providedinaconvenientsingle-tubeformat

CustomTaqMan®GeneExpressionAssaysareavailableforanyspecies,anysplicevariant,oranynovelgene.SimplyusetheCustomTaqMan®Assay

DesignTool(www.appliedbiosystems.com/cadt)toformatandsubmityourtargetsequence,ortosearchforspecificsequencesorpre-designedassays

foryourgeneofinterest.Thesoftwareeasilyguidesyouthroughtheorderingprocess,fromselectingtheassaysize,formattingyourtargetsequenceto

identifytheideallocationoftheprobe,andsubmittingyourorderviaemail.Allfilesubmissionsaredoneinasecureformattoensurethatyourtarget

sequencesandtheassociatedassaysthataredesignedremainconfidential.WithCustomTaqMan®GeneExpressionAssays,youbenefitfromApplied

Biosystems’proprietarysoftwarealgorithmsforprimerandprobedesign,whichenableyoutoobtainoptimalassaysforeachtargetsequence.Assaysare

deliveredinasingle-tube,ready-to-useformat,alongwiththeprimerandprobesequencesdesignedfromyoursubmittedsequence.

Description Concentration # of 20 µL reactions Dye labels Universal formulation Delivery time Cat. No.TaqMan® Gene Expression Assays

Inventoried 20X 250 FAM™dye Yes 3–5days 4331182

Made-to-Order 20X 360 FAM™dye Yes 5–10days 4351372

CustomTaqMan®GeneExpressionAssays

20X 360 FAM™dye Yes 10–14days 4331348

20X 750 4332078

60X 2,900 4332079

TaqMan® Endogenous Controls

Notprimer-limited FAM™dye Yes Various:seepages90,107

Primer-limited VIC®dye Yes

CustomTaqMan®Probes FAM™dyeVIC®dyeTET™dyeNED™dye

No 4–7days Various:seepage90

Custom TaqMan® Probes and Primers

→ Choiceofdyelabels,quenchers,andsynthesisscale

→ Availableforanyspeciesororganism

→ Foruseinquantitativegeneexpression,SNPgenotyping,otherallelicdiscriminationapplications,andpathogendetection

WhenyouknowtheexactsequencesyouneedforyourTaqMan®probesandprimers,AppliedBiosystemscansynthesizethemforyou(Table11.1).

Asthemarketleaderinreal-timePCR,ourhigh-qualitycustomproductscanbeusedinallyourreal-timeandend-pointapplications.Theseprod-

uctsofferyoutheidealinflexibilityifyouprefertooptimizeyourownreactionformulationorifyousimplyprefertobuyinbulk.

Table 11.1. TaqMan® probe usage chart for gene expression. Forgeneexpression,theminimumnumberofreactionsobtainedfromourTaqMan®probeprod-uctswerecalculatedbasedonuniversalassayconditions,primerconcentrationsof900nM,andprobeconcentrationsof250nM.Thenumbersareshownfor50μLand20μLreactionvolumes.

TaqMan® probe quantity Number of 50 µL reactions (96-well plates) Number of 20 µL reactions (384-well plates)

6,000pmol 480 1,200

20,000pmol 1,600 4,000

50,000pmol 4,000 10,000

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qRT-PCR directly from cells

Cells-to-Ct™ family of kitsReadytoperform.Rightoutofthebox.

→ Complete,pre-optimizedexpressionworkflowsinasingleboxarerobustandreliable

→ Preparesamplesatroomtemperaturein10minutesorless,includingDNasetreatment

→ ResultsequivalenttopurifiedRNAwithvalidatedaccuracy,reproducibility,andsensitivity

→ ValidatedwithhundredsofprimerssetsandTaqMan®GeneExpressionAssays

Cells-to-Ct™Kitsenableyoutoquicklyandeasilytransformculturedcellsintoreal-timePCRresults.AbreakthroughcelllysisandRNAstabiliza-

tiontechnologyeliminatestheneedforRNApurification.Becausesamplescanbeprocesseddirectlyincultureplates(96-or384-well),sample

handlingandthepotential forsamplelossortransfererrorareminimized,resultinginhigherreproducibility.Cells-to-Ct™Kits,however,don’t

stopatsamplepreparation.Thelysistechnologyisintegratedintoacompleteworkflowthatincludesreversetranscriptionreagentsandhigh-

performanceTaqMan®-orSYBR®Green–basedPCRmastermixes(Figure11.2).

Formoreinformation,visitwww.invitrogen.com/cellstoct.

Product Delivery time Quantity Cat. No.TaqMan®TAMRA™Probes 4–5days 6,000pmol* 450025

4–5days 20,000pmol* 450024

4–5days 50,000pmol* 450003

TaqMan®Probesareavailablewithachoiceof5'fluorescentlabel—6-FAM™,VIC®,orTET™dye*—and3'QuencherTAMRA™probe.AllprobesareHPLC-purifiedandsequence-verifiedbymassspectrometry.

TaqMan®MGBProbes 6–7days 6,000pmol* 4316034

6–7days 20,000pmol* 4316033

6–7days 50,000pmol* 4316032

TaqMan®MGBprobesareavailablewithachoiceof5'fluorescentlabel—6-FAM™,VIC®,TET™,*orNED™dye**—anda3'minorgroovebinder(MGB)/nonfluorescentquencher(NFQ).AllprobesareHPLC-purifiedandsequence-verifiedbymassspectrometry.ForResearchUseOnly.Notforuseindiagnosticprocedures.

*Pleasenotethatfilter-basedinstrumentssuchastheABIPRISM®7000SequenceDetectionSystemandAppliedBiosystems®7300/7500/7500FastReal-TimePCRSystemsarenotsuppliedwithcalibrationplatesforTET™dye.TheseinstrumentsmaybecustomcalibratedtouseTET™dyeinasingleplexreaction,butTET™dyeshouldnotbeusedinamultiplexreactionwitheitherFAM™orVIC®dyes,astheTET™dyewillnotbedistinguishedbytheseinstruments.**PleasenotethattheAppliedBiosystems®7500Real-TimePCRSystemisoptimizedforusewithNED™dye–labeledprobes.ProbeslabeledwithNED™dyewillgivelowersignalintensitiesonotherreal-timeinstrumentsystemsthanprobeslabeledwith6-FAM™,VIC®,orTET™dye.3'label:MGBNFQ(minorgroovebinder/nonfluorescentquencher).

Figure 11.2. Cells-to-Ct™ Kits provide a complete workflow from cells to qRT-PCR. Cells-to-Ct™Kitsrequire10minorlesstoreleasenucleicacidsintoacelllysatesolutionatroomtemperaturethatiscompatiblewiththeincludedreversetranscriptaseandreal-timePCRreagents.

1. Wash cells with PBS2. Add Lysis Solution and incubate for 5 minutes3. Add Stop Solution and incubate for 2 minutes

1. Mix lysate with RT master mix2. Run the RT thermal cycle for 65 minutes

1. Mix real-time PCR master mix with cDNA2. Run the PCRs in the appropriate standard or Fast real-time PCR instrument

5'

5'

5'3'

3'3'

cDNA5'3'

mRNART primer

7 min total

1. Cell Lysis 2. Reverse Transcription (RT) 3. Real-Time PCR

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Protein separation and western blotting

Toverifyhowknockdownofageneresultsinphenotypicdifferences,analyzeproteinfromRNAi-treatedcellsbyproteinseparationandwest-

ernblottingtechniques.Invitrogensupplieskits,reagents,andservicesforproteinseparation,stains,standards,blotting,anddetectionmeth-

ods.PopularproductsincludetheNuPAGE®gelsystem,SimplyBlue™SafeStain,MagicMark™XPWesternProteinStandard,andWesternBreeze®

ImmunodetectionKits.

TheNuPAGE®Novex®precastgelsystemisarevolutionaryhigh-performancepolyacrylamidegelsystem.TheNuPAGE®systemconsistsof

NuPAGE®Novex®Bis-Trisgelsandbuffersforsmalltomid-sizedproteins,andNuPAGE®Novex®Tris-acetategelsforlargerproteins.Theunique

formulationandneutraloperatingpHofNuPAGE®gelsduringelectrophoresisoffersignificantadvantagesoverothergelsystems:

→ Longestshelflife—upto1year

→ Bestresolution,sharpestbands

→ Fastestruntimesandmostefficienttransfers

→ Highestproteincapacityandgreatestproteinstability

Foracompletelistofproductsorcustomproteinservices,visitwww.invitrogen.com/proteomics.

Antibodies and immunodetection

Invitrogen’simmunodetectionofferingsincluderesearchandpathologyantibodiesandflowcytometry,cytokineandsignaling,andlabelingand

detectionproducts.Findhigh-qualityantibodies,assays,andkitsforimmunodetectionatwww.invitrogen.com/antibodies.

Nucleic acid purification and quantification

Invitrogen’snucleicacidpurificationtechnologies, formats,andkitshelpmeet thechallengesposedbyparticularnucleicacid types, sample

sourcesandvolumes,throughput levels,anddownstreamapplications.Featuredproducts includetheTRIzol® lineofproductsandPureLink™

DNAandRNApurificationsystems.ForquantifyingDNA,RNA,andproteins,Quant-iT™technologyisthemostaccuratemeans.Quant-iT™tech-

nologyusessensitivedyesthatbecomehighlyfluorescentuponbindingtotheirtargets.Unlikeassaysthatrelysolelyonabsorbance,assaysusing

thesedyesareextremelyselectiveforthemoleculebeingquantified.Ourproductsfornucleicacidpurificationandquantificationcompriseone

ofthemostcomprehensivecollectionsavailable:

→ Technologies—reagents,filtercolumns,plates,andbeads(magneticandnonmagnetic)

→ Formats—manual,high-throughput,andautomatable

→ Sampletypes—cells,blood,andtissue,includingformalin-fixed,paraffin-embedded(FFPE)forensicsamplesandmore

→ Macromolecules—purificationandquantificationofDNA,RNA,andprotein

Visitwww.invitrogen.com/napforacompletedescriptionofnucleicacidpurificationandquantificationtechnologies.

Gene expression microarray analysis

DNAmicroarraysarethemostcommonlyusedmethodfordetectingglobalchangesingeneexpressionacrossthetranscriptome. Invitrogen

suppliesacompletelineofsamplelabeling,microarraycontent,qualitycontrol,andhybridizationproducts.Extensivelycitedproductsinclude

theSuperScript®PlusDirectandIndirectcDNALabelingSystems,theSuperScript®RNAAmplificationSystems,andtheSuperScript®One-Cycle

cDNAKit(forAffymetrix®One-Cycleassays).Formoreinformation,visitwww.invitrogen.com/microarray.

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CHAPTER 12

RNAi servicesRNAi molecules for in vivo applicationsTheobstaclesandchallengesforin vivoRNAideliveryareverydifferentfromthoseinin vitrosettings.Toachievesuccessfulknockdown,in vivo

siRNAhastosurviveopsonizationanddegradationbynucleases,targetparticularcells,andtrafficintotheappropriatecellcompartment.This

chapterisdesignedtoprovideyouwiththeguidelinesandprotocolsforsuccessfulin vivoRNAiexperiments.

RNAi services

Access years of scientific experienceStrategicoutsourcingcandramaticallyincreasethepaceofdiscoveryanddecreasethecostofproductdevelopment.Anoutsourcingpartner

contributesspecializedscientificexpertiseandstate-of-the-arttechnology—whetherinmolecularbiology,RNAidelivery,cloningandviralpro-

duction,ordevelopmentofcell-basedorbiochemicalassays.Allowingresearcherstofocustheireffortsondeterminingthebesttargetsandcom-

poundstopursue,Invitrogen’sscientistsprovidetheexpertiserequiredtotakeaprojectfrombeginningtoendinatimely,professionalmanner.

Withabroadportfolioofproductsandhigh-qualityservices, Invitrogen is the industry leader in thepowerfulnewtechnologyofRNAi.

Invitrogen‘sCustomRNAiServicesutilizes syntheticandvectorRNAi technologiesanddownstreamassays tohelpcustomersachieve robust

knockdownandtofurthertheoverallbreadthofknowledgeofcomplexbiologicalsystems.RNAiServicesoffersmanyadvantages:

→ State-of-the-arttechnologies,equipment,andhigh-throughputcapabilities

→ QualityRNAireagentswithprovenperformance—StealthRNAi™siRNA,BLOCK-iT™siRNA,BLOCK-iT™shRNAvectors,andBLOCK-iT™PolII

miRRNAivectors

→ Anexpertscientificteamwithyearsofexperienceingeneknockdownstudies

→ Fast,reliableresultsthatarecompetitivelypriced

→ Professionalcustomersupportandconsulting

EachRNAiServicesprojectcanbecustomizedtofitspecificscientificgoals(Figure12.1).WhetheryouneedassistanceindesigningRNAireagents

or you desire a more expansive service such as high-throughput cloning, screening, or lentiviral production, Invitrogen has the resources to

accelerateresearch.

Cloning of RNAi vectors miR RNAi or shRNA

RNAi synthesis Stealth™ RNAi or siRNA

Functional validation

Functional validation

Sequence transferor chaining

Project consultation Experimental design

Sequence design

Lentiviral and adenoviral production

Phenotypic assay development and

screening

miRNA pro�ling

Delivery optimization

Figure 12.1. Invitrogen RNAi Services workflow.

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RNAi design services

Successful RNAi experiments start with well-designed molecules. Invitrogen’s RNAi Design Services uses a proprietary algorithm to generate

highlyeffectiveStealthRNAi™siRNA,BLOCK-iT™siRNA,BLOCK-iT™PolIImiRRNAi,andBLOCK-iT™shRNAsequences.Thisservicecanaccommo-

dateavarietyofsequencerequests:

→ DesignoflargenumbersofRNAisequencesforoneornumerousgenes

→ Designofsequencestotargetdifferentsplicevariantsormultiplespecies

→ ConversionofsiRNAtoStealthRNAi™siRNAsequences,andofStealthRNAi™siRNAtoBLOCK-iT™PolIImiRRNAisequences

→ Designofsequencesforcreatingreportervectors

→ DesignofLUX™FluorogenicPrimersforevaluatingRNAireagentsbyquantitativereversetranscriptionPCR(qRT-PCR)

Stealth RNAi™, Silencer® Select, and Silencer® siRNA custom synthesis

SyntheticallygeneratedRNAimoleculessuchasStealthRNAi™,Silencer®Select,andSilencer®siRNAarethemostpopularRNAimoleculesusedin

experiments.Theseduplexesareeasilysynthesizedwithinjustafewdays.WeofferCustomRNAiSynthesisServicesforStealthRNAi™,Silencer®

Select,andSilencer®siRNAmolecules,butwerecommendtheuseofchemicallymodified,blunt-ended25-merStealthRNAi™double-stranded

duplexes. Stealth RNAi™ siRNA reduces off-target effects, avoids induction of stress response pathways, and has enhanced nuclease stability

withoutalossinpotency.

Vector-based RNAi services

DNA-based or vector-mediated RNAi is often used for long-term expression, hard-to-transfect cell lines, tissue-specific RNAi expression, and

inducibleRNAi.TheDNAvectorsexpressRNAsequencesthatareprocessedbyDicer,anendogenousenzyme,intofunctionalRNAiduplexesthat

leadtoknockdownofthetargetmessage.Vectortechnologieshelpaccomplishthesegoals:

→ Achievetransientorstabletargetknockdown

→ PerformRNAiinanycelltype—evenhard-to-transfect,primary,andnondividingcells

→ RegulategeneinhibitionwithinducibleRNAi

→ Enablestudyoflong-termgeneknockdown

→ TrackexpressionofRNAisequenceswithEmeraldGreenFluorescentProtein(EmGFP)

→ Targetmultiplegenesforknockdownusingonevector

Invitrogen’s RNAi vector technologies take advantage of the Gateway® system for easy cloning, and include both BLOCK-iT™ Pol II miR RNAi

vectorsandBLOCK-iT™shRNAvectors.Chapter8containsmoredetailson the featuresandbenefitsof the twovector technologiesand the

Gateway®system.

Cloning of BLOCK-iT™ Pol II miR RNAi vectorsBLOCK-iT™PolIImiRRNAivectorscombinethebenefitsoftraditionalshRNAvectors—stableexpressionandtheabilitytouseviraldelivery—with

capabilitiesfortissue-specificexpressionandmultiple-targetknockdownfromthesametranscript.TheseBLOCK-iT™PolIImiRRNAisequences

canbedesigned,synthesized,andthenclonedintooneofthefollowingmiRRNAientryvectors:pcDNA™6.2-GW/miRorpcDNA™6.2-GW/EmGFP-

miR (seeChapter8 formore informationonthesevectors).EachmiRRNAientryvectorhasaPol IIcytomegalovirus (CMV)promoter todrive

expression,attBsitesforcompatibilitywithGateway®technologyandsimplifiedcloning,andtheabilitytobeexcisedfromalongPolIItranscript

for expression of more than one miR RNAi knockdown sequence from the same vector. In addition, the pcDNA™6.2-GW/EmGFP-miR vector

featurescocistronicexpressionof theEmGFPreporter, so thatEmGFPexpressioncanbecorrelatedwith theknockdownactivityof themiR

RNAi vector expression cassette. High-quality sequenced BLOCK-iT™ Pol II miR RNAi vectors are delivered as glycerol stocks and are ready

fortransfection.

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Chaining BLOCK-iT™ Pol II miR RNAi sequencesTheBLOCK-iT™Pol IImiRRNAivectorsarecapableofexpressingmorethanonemiRRNAisequenceonthesametranscript,allowingfor the

knockdownofmultiplegenessimultaneously.Usingourrestrictionenzymeprocedure,wecanlink,inanyorder,twoormoreclonedmiRRNAi

sequencesfromthepcDNA™6.2-GW/miRorpcDNA™6.2-GW/EmGFP-miRentryvector.WedeliverthefinalvectorscontainingthechainedmiR

RNAi sequences as glycerol stocks that can be prepared for transfecting cells or subcloning into a destination vector for a variety of down-

streamapplications.

Subcloning BLOCK-iT™ Pol II miR RNAi vector sequencesThroughGateway®recombination,experimentalpossibilitiesaregreatlyincreased.ThemiRRNAicassette,whichcontainsEmGFP(pcDNA™6.2-GW/

EmGFP-miRvectoronly),miRflankingregions,andmiRRNAisequencehomologoustothetargetofinterest,canbetransferredtomanydestina-

tionvectors.Availabledestinationoptionsincludethefollowing:

→ Lentiviralvectorsforstabletransductionofdividingandnondividingcells

→ Vectorswithtissue-specificorregulatedpromoters

→ Vectorswithalternativereporterfusionconstructs

→ Flp-In™systemforsingle-sitechromosomalintegration

The BLOCK-iT™ Pol II miR RNAi Subcloning Service includes the Gateway® BP and subsequent LR recombination reactions to move the miR

RNAicassette fromthemiRRNAientryvector into theGateway®destinationvectorofchoice.The resultingvectorsaredeliveredasglycerol

stocks that can be prepared for transfection or used to produce lentiviral particles. For a list of Invitrogen’s destination vectors, please visit

www.invitrogen.com/gateway.

Cloning BLOCK-iT™ shRNA vectorsIt is important to begin knockdown experiments with high-quality, error-free shRNA vectors. BLOCK-iT™ shRNA sequences can be designed,

synthesized,andclonedintooneoftwoshRNAentryvectors:pENTR™/U6orENTR™/H1/TO(seeChapter8formoreinformationonthesevectors).

EachshRNAentryvectorhasaPolIIIpromotertodriveexpressionandattLsitesforcompatibilitywithGateway®technologyandsimplifiedclon-

ing.WecanalsomovetheRNAiexpressioncassettetoanumberofothervectorbackbonesforexpandedexperimentalchoices.Thesequenced,

verifiedBLOCK-iT™shRNAvectorsaredeliveredasglycerolstocksreadyfortransfectionoradenoviralorlentiviralproduction.

Subcloning BLOCK-iT™ shRNA sequencesForincreasedexperimentalflexibility,theshRNAcassettecontainingthePolIIIpromoterandanshRNAsequencehomologoustothetargetof

interestcanbetransferredtomanyBLOCK-iT™destinationvectorsthroughaGateway®LRrecombinationreaction.Currently,fiveBLOCK-iT™DEST

vectors,includinglentiviralandadenoviralvectors,areavailable.Theresultingvectorsaredeliveredasglycerolstocksthatcanbepreparedfor

transfectionorusedtoproducelentiviraloradenoviralparticles.

Delivery optimization services

Achieving robust gene inhibition begins with efficient delivery of RNAi reagents while minimizing toxicity. Optimizing the RNAi transfection

conditionsforaparticularcell lineisthefirststepinanyRNAiexperiment,andwehavebeenoptimizingtransfectioninabroadrangeofcell

linesforoveradecade.Foreasy-to-transfectcelllines,wetypicallyusecationiclipidtransfectionreagents.Fordifficult-to-transfectcelllines,we

recommendviraldelivery.IntheDeliveryOptimizationServices,weuseourknowledgeofandexpertisewithviralvectorsandnonviralreagents

totestamatrixofdeliveryparametersandfindtheoptimaldeliveryconditionsforthecelllineofinterest.

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Viral production

Manymammaliancelltypesandexperimentalsituationscreatechallengesforstandardtransfectionexperiments.Lentiviralandadenoviralsys-

temsallowviraldeliveryofBLOCK-iT™PolIImiRRNAivectorsandBLOCK-iT™shRNAvectorstovirtuallyanycelltype:

→ Postmitoticandnondividingcells

→ Primarycells

→ Stemcells

→ Growth-arrestedcells

BLOCK-iT™ lentiviral production servicesLentiviralparticlescontainingBLOCK-iT™PolIImiRRNAiorBLOCK-iT™shRNAsequencesareactivelyimportedintothenucleiofcellsbycis-actingele-

ments.ExpressionofRNAisequencesofinterestcanoccurtransiently,butafterintegrationintothegenome,expressionofsequencesisstable,resulting

incontinuoushighyieldsofexpression.Invitrogen’sViralProductionServicescreatesreplication-incompetentlentiviralparticlesthatefficientlytransduce

nearlyanydividingornondividingcelltype.Crudelentiviraltiterstypicallyrangefrom104to106transducingunitspermilliliter(TU/mL)asmeasuredbya

blasticidinresistanceassay,andtheworkinglentiviralstocksareavailableintwoscalestomeetindividualexperimentalneeds:50or100mL.Largersizes

maybeavailable.TheresultingtiteredlentiviralstocksareshippedreadyforuseinRNAiknockdownexperiments.

When experiments require lentiviral stocks with titers above 104–106 TU/mL, Invitrogen can concentrate 50–100 mL of crude lentivirus

downto1–2mLofconcentratedviralstock.Thisprocesswillincreasetheconcentrationofthestock25-to100-fold.Thefinallentiviralstocksare

shippedreadyforuseinRNAiknockdownexperiments.

BLOCK-iT™ adenoviral production servicesAdenoviral systemsarepopularplatforms for reliabledeliveryandhigh-level transientexpressionofBLOCK-iT™Pol IImiRRNAiorBLOCK-iT™

shRNAsequencesinanymammaliancelltype.Invitrogenusesa293Aproducercelllinetogeneratehightitersofreplication-incompetent(E1-and

E3-deleted)adenoviralparticlesthatdeliverandexpressRNAisequences.Crudeadenoviraltiterstypicallyrangefrom108to109plaque-forming

unitspermilliliter (PFU/mL),and theworkingadenovirus stocksareavailable in threescales:10,50,or100mL.Larger sizesmaybeavailable.

TheresultingtiteredadenoviralstocksareshippedreadyforuseinshRNAknockdownexperiments.Forsomeapplications,itmightbeneces-

sarytostartwithadenoviral titersabovethenormal108to109PFU/mLthataretypicallyachievedwiththeBLOCK-iT™AdenoviralProduction

Services.WiththeBLOCK-iT™AdenoviralConcentrationServices,10–100mLofcrudeadenoviruscanbeconcentratedtoachievetiters inthe

1010–1011PFU/mLrange.ThefinaladenoviralstocksareshippedreadyforuseinRNAiknockdownexperiments.

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RNAi functional validation services

InvitrogencanrapidlyidentifyeffectiveRNAireagentsthatcanbeusedinfurtherfunctionalstudies.UnderstandingthatthegoalofeachRNAi

experimentisunique,InvitrogenoffersawidearrayofapproachestoscreenforthemosteffectiveRNAireagents.

Quantitative RT-PCR (qRT-PCR)—measure mRNA knockdownThismethodsensitivelymeasuresendogenousmRNAlevels.Ifthetargetgeneisexpressedatareasonablelevelinthespecifiedcellline,qRT-PCR

isfastandreliable.

RNAi target screening system—use a reporter-based system for preliminary testsThismethodispreferredforhigh-throughputapplicationsortoscreenforeffectiveRNAireagentsbeforemovingexperimentsintoadifficultcell

line.TheStealthRNAi™siRNAorsiRNAduplexiscotransfectedwiththepSCREEN-iT™/lacZreportervector,andβ-galactosidaselevelsareusedto

quantitativelydeterminetheeffectivenessoftheRNAiduplex.

Western blot analysis—measure protein knockdownThismethodisbestforclearlyshowingthatlossoffunctionisduetospecificendogenousproteinknockdown,andremovesconcernsthatprotein

half-lifeisaffectingphenotype.

Dose response/IC50 assays—determine optimal conditions for a chosen systemDoseresponsedatashowthedynamicrangeofRNAisuppressionandenablethemostappropriatesiRNAconcentrationtobeselectedforacho-

sencelltype.IC50istheconcentrationofthesiRNAduplexthatisrequiredfor50%inhibitionofthetargetandiscommonlyusedasameasureof

drugeffectiveness.Thedoseresponseisalsoagoodwaytodetermineconditionsthatshowoptimalknockdownandminimaltoxicityassociated

withthedeliverymethod.InvitrogencanevaluatesiRNAduplexesatvarioustimepointsinaparticularcellline.

Inducible RNAi—screen with RNAi expression “on” and “off”BothBLOCK-iT™PolIImiRRNAiandBLOCK-iT™shRNAvectorscanbeclonedforinducibleRNAiwithtighttetracyclineregulation.

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Phenotypic assay development and high-throughput screening services

Theultimategoalofgeneknockdownistoobserveachangeinphenotype.HavingmanyyearsofexperiencewithRNAiexperimentsandphe-

notypicassays,Invitrogenwillusethisexperienceandknowledgetohelpdevelopandconductassaystailoredtospecificresearchprojects.The

RNAiservicesfocusondeliveringthemostreliabledatautilizingthemostappropriateRNAitechnologyforphenotypicassays.Awiderangeof

phenotypic assays can be performed for a variety of target classes—kinases, phosphatases, proteases, nuclear receptors, G-protein–coupled

receptors(GPCRs),ionchannels,andcytochromeP450s—includingassaysofthefollowingtypes:

→ Morphological

→ Enzymatic

→ Biochemical

→ Cell-based

→ Customized

Custom stable cell line generationInvitrogenCustomServicescantransformacelllinetostablyexpressanRNAiknockdownsequenceorgeneofinterestataconstantlevel,indefinitely.

Thehomogeneouscellpopulationexpressingtheknockdownsequenceortargetgenecanbeusedformanyexperiments,includingdifferentiation

studies,inducibleexpressionstudies,andin vivotransfer.Invitrogen’sscientistswillassistyouincreatingastablecellline,andperformqualitycontrol

testingtoensurethatthecelllinemeetsrequirements.ThesearesomeoftheadvantagesofhavingInvitrogencreateyourstablecellline:

→ Skilledscientistswithspecializedtrainingincreatingstablecelllines,alongwithexpertcustomersupportandconsulting

→ Widerangeofqualitycontroltestingassays

→ BLOCK-iT™PolIImiRRNAiandBLOCK-iT™shRNAvectortechnologiesandservices

→ GenerationofT-REx™hostcelllinesforexpressionthatcanberegulated

→ Gateway®cloningservicesforageneofinterest

InvitrogencancreatecelllinesbytransfectionofaDNAplasmidandsubsequentselection,orbyusinglentiviralparticles.Lentiviralparticlescan

integrateDNAefficientlyintotherecipientcellgenomeandcanreducethetimelineforcreatingthestablecellline.ThegeneofinterestorRNAi

sequencewillberandomlyintegratedintothecell’sgenome.Tofindatleastoneclonethatmeetsyourexpressionrequirements,Invitrogenwill

expandandtestmanystableclones.Thefinalstablecelllinewillbetestedformycoplasmasandsuppliedreadyforfurtherexperiments.

RNAi custom collaborative research

Usingafullycollaborativeapproach,Invitrogencanworkwithyoutocustom-designandexecuteanRNAiapproachtofityourneeds.Fromsimple

customassaystolong-termarrangementsfortargetdiscoveryandvalidation,Invitrogenhasthehistory,experience,andtechnologytohelpyou

succeed.Giveusacalltodaytodiscussyourvision.

NOTE: Find information on RNAi services online at www.invitrogen.com/customservices.

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CHAPTER 13

Overview of miRNAsAsthecomplexityofagenomeincreases,sodoestheratioofnon-codingtocodingRNAs[1].Non-codingRNAsappeartobeinvolvedinavariety

ofcellularroles,rangingfromsimplehousekeepingfunctionstocomplexregulatoryfunctions.Ofthevarioussubclassesofnon-codingRNAs,

microRNAs(miRNAs)arethemostthoroughlycharacterized.Thesesingle-strandedRNAsaretypically19to22nucleotideslongandarethought

toregulategeneexpressionposttranscriptionallybybindingtothe3´untranslatedregions(UTRs)oftargetmRNAsandinhibitingtheirtranslation

[2].RecentexperimentalevidencesuggeststhatthenumberofuniquemiRNAsinhumanscouldexceed1,000[3],thoughseveralgroupshave

hypothesizedthattheremaybeupto20,000[4,5]non-codingRNAsthatcontributetoeukaryoticcomplexity.

miRNA function and regulationBothRNApolymeraseIIandIIItranscribemiRNA-containinggenes,generatinglongprimarytranscripts(pri-miRNAs)thatareprocessedbythe

RNaseIII–typeenzymeDrosha,yieldinghairpinstructures70to90basepairs in length(pre-miRNAs).Pre-miRNAhairpinsareexportedtothe

cytoplasm,wheretheyarefurtherprocessedbytheRNaseIIIproteinDicerintoshortdouble-strandedmiRNAduplexes19to22nucleotideslong.

ThemiRNAduplexisrecognizedbytheRNA-inducedsilencingcomplex(RISC),amultiple-proteinnucleasecomplex,andoneofthetwostrands,

theguidestrand,assiststhisproteincomplexinrecognizingitscognatemRNAtranscript.TheRISC-miRNAcomplexofteninteractswiththe3´

UTRoftargetmRNAsatregionsexhibitingimperfectsequencehomology,inhibitingproteinsynthesisbyamechanismthathasyettobefully

elucidated(Figure13.1).

PlantmiRNAscanbindtosequencesontargetmRNAsbyexactornear-exactcomplementarybasepairingandtherebydirectcleavageand

destructionofthemRNA[6,7].SimilartothemechanismemployedinRNAinterference(RNAi),thecleavageofasinglephosphodiesterbondon

thetargetmRNAoccursbetweenbases10and11[8].Incontrast,nearlyallanimalmiRNAsstudiedsofardonotexhibitperfectcomplementar-

itytotheirmRNAtargets,andseemtoinhibitproteinsynthesiswhileretainingthestabilityofthemRNAtarget[2].Ithasbeensuggestedthat

transcriptsmayberegulatedbymultiplemiRNAs,andanindividualmiRNAmaytargetnumeroustranscripts.Researchsuggeststhatasmanyas

one-thirdofhumangenesmayberegulatedbymiRNAs[9].AlthoughhundredsofmiRNAshavebeendiscoveredinavarietyoforganisms,little

isknownabouttheircellularfunction.SeveraluniquephysicalattributesofmiRNAs,includingtheirsmallsize,lackofpolyadenylatedtails,and

tendencytobindtheirmRNAtargetswithimperfectsequencehomology,havemadethemelusiveandchallengingtostudy.

iDrosha

AAAAAA

Primary miRNA(pri-miRNA)

Precursor miRNA(pre-miRNA)

Dicer

Nuclearpore

Xpo-5

miRNA

Unwinding

Nucleus

CytoplasmmiRNA has 100% homology to mRNA,which results in target mRNA cleavage

RISC

Pol II and Pol IIImRNA transcription

miRNA gene

m7Gppp

miRNA binds 3´ UTR with imperfect homology, inhibiting translation

RISC

3´ UTR

Figure 13.1. Biogenesis and function of miRNA. MicroRNA transcripts, generated by RNA polymerases II and III, are processed by the RNase III enzymesDrosha(nuclear)andDicer(cytoplasmic),yielding19–22nucleotidemiRNAduplexes.OneofthetwostrandsoftheduplexisincorporatedintotheRISCcomplex,whichregulatesproteinexpression.

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Which miRNA profiling or validation system should I use?

Figure 13.2. Which miRNA profiling system should I use?

Figure 13.3. Which miRNA validation system should I use?

TaqMan® Assays or NCode™ platform?

I have a small amount of sample

I need precise quantitation of levels

of miRNAs

Do you have access to an AB 7900HT?

Do you have access to an AB 7900HT?

I am not working in human, mouse,

or rat

We recommend NCode™ miRNA

Microarrays

We recommend the Megaplex™ primer pool

work�ow utilizing TaqMan®

Assay technology

We recommend the Megaplex™ primer pool

work�ow utilizing TaqMan®

Assay technology

YES YES

TaqMan® Assays or NCode™ platform?

I want to be very speci�c in my analysis

I need precise quantitation of levels

of miRNAs

I want to quantitate mRNA and miRNA in

the same sample

Do you have access to an AB instrument?

Do you have access to an AB instrument?

We recommend NCode™ miRNA qRT-PCR Assays

We recommend TaqMan® MicroRNA Assays or

Custom TaqMan® Small RNA Assays

We recommend TaqMan® MicroRNA Assays or

Custom TaqMan® Small RNA Assays

YES YES

13Overview of m

iRNAs



Genome-wide small RNA discovery and profiling

UsetheSOLiD™SystemtodiscovernovelsmallRNAsandsimultaneouslydeterminewhichmiRNAsareexpressed,andwhereandwhentheyare

expressed.

MicroRNA Profiling Using TaqMan® Chemistry-Based TechnologyUseMegaplex™PrimerPoolsandTaqMan®MicroRNAArraystofindoutwhichmiRNAsareexpressed,andwhereandwhentheyareexpressed.

Targeted MicroRNA QuantitationUseindividualTaqMan®MicroRNAAssaystogetspecificinformationaboutwhereandwhenmiRNAsareexpressed.

MicroRNA Functional AnalysisUsesyntheticmiRNAmimicsandinhibitorsforgain-andloss-of-functionstudiestolearnwhichcellprocessesareregulatedbymiRNAs.Further

investigatemiRNAtargetsandeffectsonproteinexpressiontodeterminewhichgenesmiRNAscontrol.

MicroRNA: a new frontier in biology

MicroRNAs (miRNAs) are small, non-coding RNA molecules that direct posttranscriptional suppression of gene expression. The mechanisms

by which miRNAs exert their regulatory effects are still being investigated, but it is well established that they interact with messenger RNAs

(mRNA)basedonsequencehomologyandultimatelyaffecttranslationand/orstabilityofthetargetedmRNA.EachmiRNAmayregulatemultiple

genes,anditislikelythatmorethanonethirdofallhumangenesmayberegulatedbymiRNAmolecules[10].Firstreferredtoasthe“biological

equivalentofdarkmatter”[11],miRNAshavebeenshowntoactaskeyregulatorsinmanybasicbiologicalprocessessuchasdevelopment,cell

proliferation,differentiation,andthecellcycle.EmergingevidencealsoimplicatesmiRNAsinthepathogenesisofhumandiseasessuchascancers,

metabolicdiseases,neurologicaldisorders,infectiousdiseases,andotherillnesses[12].

Fundamental questions in microRNA research

ResearchershaveonlyjustbeguntounderstandhowmiRNAsworkandwhatrolestheyplayinbiology.Thus,evenfundamentalquestionsabout

miRNAshaveyettobeanswered.LaboratoriesaroundtheworldareexaminingtheeffectsofmiRNAs invariousexperimentalsystems. Initial

inquiriestypicallyinvestigatewhichmiRNAsarepresentinaparticularcelltypeandthedifferencesinmiRNAexpressionbetweendifferenttis-

sues,developmentalstages,ordiseasestates.Additionally,manyeffortstodiscoverpreviouslyunreportedmiRNAsareunderway.OncemiRNAs

ofinterestareidentified,subsequentstudiesmaybedesignedtoartificiallyperturbmiRNAexpressionlevelsinculturedcellswhilemeasuring

resultingmRNA,protein,orphenotypicchanges.Ultimately,thegoalofmanyprojectsistoidentifytheeffectsofmiRNAsoncellularprocesses,

andasaresult,assesstheirbiologicalimpactandsignificance.

Invitrogen and Applied Biosystems, both part of Life Technologies, lead the way in developing microRNA research tools

Active,maturemiRNAsaretypically17–24nucleotide,single-strandedRNAmoleculesthatareexcisedfromlargerprecursors.Becauseoftheir

smallsize,thestudyofmaturemiRNAandotherclassesofsmallRNArequiresspecializedtechniques.InvitrogenandAppliedBiosystemsprovide

innovativetoolsdevelopedspecifically formiRNAresearch, fromisolationthroughdiscovery,profiling,quantitation,validation,andfunctional

analysis.ThesetoolsenablescientiststonotonlyaddressthefundamentalquestionsaboutmiRNA,buttousetheseadvancestorealizethefull

potentialofthisnewandexcitingchapterinbiologicalscience.

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Capture the sequence and expression level of every small RNA in your sample

ThediscoveryofnewsmallRNAsisongoing;2,000newsequenceshavebeenaddedtothemiRBaseRegistryinthelasttwoyears[13].TheApplied

BiosystemsSOLiD™RNAAnalysisSolutionincludesasuiteofproductsthatenablerapiddiscoveryofpreviouslyunreportedsmallRNAs,sensitive

quantitationofexpressionlevels,andforthefirsttime,theabilitytoassessallele-specificexpressionpatterns.

UnlikeotherdetectionmethodsthatonlyassayknownorpredictedsmallRNAsequences,theSOLiD™Systemprovidesadigitalreadoutof

thesmallRNAmoleculesinaparticularsamplewithoutpriorsequenceinformation.ThiscapabilityenablesboththediscoveryofnovelsmallRNA

molecules,andthedetectionofdifferentisoformsgeneratedbyalternativeprocessing[11].

Genome-wide small RNA discovery and profiling workflow

Featuresoftheworkflow:

→ AbilitytodiscovernovelsmallRNAmoleculesandisoforms

→ SensitivitytodetectsmallRNAspresentatlessthanonecopypercell

→ Orientationinformationispreservedforstrand-specificexpressionanalysis

→ Enablecost-effectiveanalysisofmultiplesamplesinasinglerunusingbarcodes

How it worksSmallRNAsareisolatedfromthesampleandreversetranscribedtomakeacDNAlibraryofthesmallRNApopulation.ThecDNAinthelibraryis

thenamplifiedandsequenced,generatingsequencefragments,commonlycalled“tags”,correspondingtothecDNAofeachsmallRNA.Thetags

arethenmappedbacktoreferencesequencedatabasestoidentifybothnewandknownsmallRNAspecies.Relativeexpressionlevelscanbe

calculatedbasedonthenumberoftagsobtainedforeachsmallRNA(Figure13.4).

• Ambion® mirVana™ miRNA Isolation Kit • SOLiD™ Small RNA Expression Kit • SOLiD™ ePCR Kits• Thermal Cyclers• SOLiD™ Sequencing Kits• SOLiD™ System

• Ambion® Pre-miR™ Precursors and Anti-miR™ Inhibitors• TaqMan® MicroRNA Assays • Custom TaqMan® Small RNA Assays (Early Access)

~18-100bp

inserts

MicroRNA

P1 P2IA2

Barcode

ATAA GA CA AATATT CT GT TT CA

GTGCCG

3'

1 2 3 4 5 6 7 ... (n cycles)Ligation cycle

Perform Genome-Wide Sequencing Validate ResultsPrepare Small RNA LibraryIsolate RNA

Q F

Reverse Transcription

Real-Time PCR

Stem-loop RT primer

Target Sequence

Forward Primer

TaqMan® ProbeReverse Primer

Figure 13.4. Genome-wide small RNA discovery and profiling workflow.

13Overview of m

iRNAs



Robust small RNA discovery and profiling using the leading next-generation sequencing platform

TheSOLiD™RNAAnalysisSolutionisarobustmethodforglobaldiscoveryandexpressionprofilingofRNA.TheSOLiD™SmallRNAExpressionKit

providesastreamlinedprotocolforlibraryconstructionenablingstrand-specificexpressionanalysisfromaslittleas10ngoftotalRNA.Itprovides

asimplemeanstoconvertRNAintoa libraryofdouble-strandedDNAmoleculesusingarobustone-dayprocedure(Figure13.5).Thisunique

processpreservestheorientationinformationoftheoriginalmoleculeandenablesstrand-specificexpressionanalysis.ThesmallRNAlibrariesare

thenfedintotheemulsionPCR(ePCR)stepoftheSOLiD™systemsamplepreparationworkflow.

TheSOLiD™Systemgeneratesover400Mmappablereadsperrun.Thislevelofthroughputallowsresearcherstoanalyzemultiplesamples

perrunwhilemaintainingthesensitivitynecessaryforquantitativeanalysisofmoleculespresentatlowlevels.Theabilitytomultiplexmultiple

samples,duetotheintegrationofbarcodesequencesduringlibrarypreparation,dramaticallyreducesthecost,time,andlaborassociatedwith

downstreamePCRandsequencingreactions.

TheSOLiD™Systemisahypothesis-generatingmethodforglobalsmallRNAdiscoveryandquantitation.Figure13.6showstheproportions

ofdatafromaSOLiD™Systemsequencingexperimentthatmappedtofourknownsequencedatabases.Approximately51%ofthetagsmapped

toknownmiRNAs,andmorethan40%ofthetagsmappedtogenomicregionsbutnottoanyknownRNAspecies.Someofthislattersetcould

representpreviouslyuncharacterizedmiRNAs.

DatafromthesequencetagsthatmappedtoknownmiRNAswereevaluatedtodeterminetherelativeexpressionofthecorresponding

miRNAsinhumanlungcomparedtoplacenta.MicroRNAsthatshowedsignificantlydifferentexpressioninthetwotissueswereselectedfordif-

ferentialexpressionanalysisusingTaqMan®MicroRNAAssays.Figure13.7showsacomparisonofmiRNAexpressiondatafromthetwomethods.

WithanRvalueof0.9,thisanalysissuggeststhattheSOLiD™platformisavalidprofilingtoolforgeneexpressionanalysis.

4. Small RNA Library Amplification (1–1.5 hr)

PCR Primer

PCR Primer

Barcode

5. Amplified Small RNA Library Cleanup and Size Selection by PAGE (3–4 hr)

Excise~105–150 bp

6. SOLiD™ Sample Preparation

SOLiD™ System: Sample is ready for emulsion PCR

(templated bead preparation)

1. Isolate RNA

Small RNAs Adaptor mix A or B

+

2. Hybridization/ligation (2 hr)

Ligation junctions

3. Reverse transcription and RNase H digestion (1 hr)

cDNA

Figure 13.5. SOLiD™ Small RNA Expression Kit protocol.

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Ribosomal RNA, tRNA, repeats, 5754136, 6%

RefSeq, 2856052, 3%

miRNA, 47648764, 51%

Human Genome, 38262313, 40%

Figure 13.6. Distribution of 94 million mappable sequence reads to known sequence databases. FourhumanRNAsamples(threefromplacentaandonefromlung)werepreparedandsequencedusingasingleSOLiD™Systemruninthequadslideconfiguration.Atotalof173Msequencetagsweregeneratedand94Mweremappedtotheindicateddatabaseswith0–1mismatches.Thesmallpercentage(9%)oftagsthatmappedtoknownrRNAs,tRNAs,repeatregions,andtheRefSeqdatabaseindicatethatlibrarypreparationusingtheSOLiD™SmallRNAExpressionKitsuccessfullyenrichedforthesmallRNAfraction.Asubsetofthesequencetagsthatmappedtothehumangenome,butnottoknownmiRNAs,couldrepresentpreviouslyuncharacterizedsmallRNAs.ThesespeciesarecurrentlybeingvalidatedasnovelsmallRNAsusingCustomTaqMan®SmallRNAAssays.

Figure 13.7. Good correlation between microRNA expression data from SOLiD™ System sequenc-ing and analysis with TaqMan® MicroRNA Assays. The log2of the fold-changebetweendif-ferentiallyexpressedmiRNAsinhumanlungandplacentasamplesobtainedbySOLiD™systemsequencingwascomparedtothesamedataobtainedbyanalysisusingTaqMan®MicroRNA Assays. The resulting R value of 0.9 indicates that there is good correlationbetweenrelativemiRNAexpressiondatafromthetwoplatforms.

log2 (fold change) SOLiD™ system sequence analysis

–10

–15

–10

–5

0

5

R=0.9

–5 0 5 10

log 2

(fold

cha

nge)

Ta

qM

an® M

icro

RN

A A

ssay

s

Profile microRNA expression in a single day using gold standard TaqMan® assay technology

ProfilingthedifferencesinglobalmiRNAexpressionamongsamplesisausefulfirststepinidentifyingspecificmiRNAsthatinfluenceabiological

process.Forexample,aresearchermightcomparethemiRNAprofilesindiseasedvs.healthytissue,compound-treatedvs.untreatedsamples,or

differentorgansfromasinglesubjectwiththeintentionofidentifyingthosemiRNAsthatareexpressedatdifferentlevelsbetweenthesample

types.SincemiRNAscaninfluencecellularfunction,evenatverylowconcentrations,andcanbeexpressedoveranextremelywiderange,miRNA

quantitationrequirestoolswithhighsensitivityandabroaddynamicrange.AppliedBiosystemsMegaplex™PrimerPools, inconjunctionwith

TaqMan®ArrayMicroRNACards,areidealforsuchexperiments.Usingthesetools,researcherscangenerateanexpressionprofilefor754,518,or

303miRNAsfromhuman,mouse,orrat,respectively,inasingleworkingdayfromaslittleas1ngofinputtotalRNA.Takingfulladvantageofthe

goldstandardsensitivity,specificity,anddynamicrangeaffordedbyTaqMan®Assaychemistry,andincorporatingAppliedBiosystems’innovative

stem-loopRTprimerdesignforPCRoftinytargets,Megaplex™PrimerPoolsprovidesignificantbenefitsovermicroarrays,whichrequireseveral

daysandhundredsofnanogramsofinputRNAtogeneratedata.

13Overview of m

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MicroRNA profiling using TaqMan® assay technologyFeaturesoftheworkflow:

→ Resultsthesameday—completeanexperimentprofilinghundredsofmiRNAsinaslittleas5hours(Figure13.8)

→ Idealforhuman,mouse,andratprofiling

→ Requiresonlyminutesampleamounts—aslittleas1ngoftotalRNAinput—makingitsuitableforFFPE,FACS,biopsy,andotherverysmall

samples

→ ComprehensivecoverageofknownmiRNAsconsistentwithSangermiRBasev14andv10,forhumanandmouse,respectively

How it worksUpto381miRNAsarereversetranscribedinasinglereactionusingMegaplex™RTPrimers,amixtureofmiRNA-specificstem-loopprimers.Next,

anoptionalamplificationstepcanbeperformedusingMegaplex™PreAmpPrimers.Thisunbiasedamplificationstepsignificantlyincreasesthe

concentrationofmiRNAsinthesampleenablingmaximumsensitivityanddetectionusingreal-timePCR.Forthefinalquantitationstep,TaqMan®

UniversalPCRMasterMixisaddedtoeachsampleandthemixturesarepipettedintothesampleloadingportsofaTaqMan®ArrayMicroRNA

Card—apreconfiguredmicrofluidiccardcontaining384TaqMan®MicroRNAAssays.Thereal-timePCRisrunontheAppliedBiosystems®7900HT

FastReal-TimePCRSystem.

Amplify cDNA(optional)

Load TaqMan® Array MicroRNA Card

Run TaqMan® Array MicroRNA Card Analyze Data

Reverse Transcribe

• Ambion® mirVana™ miRNA Isolation Kit

• Ambion® RecoverAll™ Total

Nucleic Acid Isolation Kit for FFPE

• TaqMan® MicroRNA Cells-to-CT™ Kit

• Megaplex™ RT Primers

• TaqMan® MicroRNA Reverse Transcription Kit

• Megaplex™ PreAmp Primers

• TaqMan® PreAmp Master Mix

• RT product & TaqMan®

Universal PCR Master Mix

• Applied Biosystems® 7900HT

Fast Real-Time PCR System

• Instrument Software (included)

• RQ Manager Enterprise Software (optional)

• Real-Time StatMiner® Software (optional)

Prepare Sample

10–30 min 150 min 90 min 10 min 120 min

134567 28

Figure 13.8. MicroRNA profiling workflow using TaqMan® technology.

Megaplex™ Primer PoolsWhetheryourprofilingexperimentrequiresultimatesensitivity,broadcoverage,orboth,Megaplex™PrimerPoolsoffertheflexibilitytoenable

youtoaccomplishyourresearchgoals(Figure13.9).ProvidingcomprehensivecoverageofSangermiRBasev14andv10,forhumanandmouse,

respectively,whenusedwithTaqMan®MicroRNAArrays,Megaplex™PrimerPoolsdelivertheidealmiRNAprofilingsolutionforbothhumanand

rodentspecies.

→ Megaplex™RTPrimers—DesignedtostreamlinemiRNAprofiling,Megaplex™RTPrimersarepoolsofRTprimersidenticaltothosefound

in individual TaqMan® MicroRNA Assays. Megaplex™ RT Primers are ideal for global miRNA expression profiling; just two pools provide

comprehensivecoverageoftheSangermiRBasev10databasecontent,andtheircontentmatchesthecorrespondingTaqMan®MicroRNA

ArrayHumanorRodentMicroRNACard.However,theycanalsobeusedtopreparecDNAforindividualTaqMan®MicroRNAAssays.

→ Megaplex™ PreAmp Primers—When samples are limiting or assay sensitivity is of utmost importance, Megaplex™ PreAmp Primers

significantlyhelpenhancetheabilitytodetecthumanandrodentmiRNAswithlowexpressionlevelsorfromlimitedamountsofsample.

Megaplex™ PreAmp Primers enable the generation of a comprehensive miRNA expression profile, using as little as 1 ng of input total

RNA(Figure13.10).WithcontentmatchedtotheMegaplex™RTPrimersandtheTaqMan®ArrayHumanorRodentMicroRNACards,cDNA

preparedbyreversetranscriptionwithMegaplex™RTPrimerscanbepreamplifiedusingMegaplex™PreAmpPrimersandTaqMan®PreAmp

MasterMixtouniformlyamplifyallmiRNAsthatwerepresentintheoriginalsample.

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Figure 13.9. Megaplex™ Primer Pools bring the power of real-time PCR to microRNA profiling experiments.

Megaplex™ PreAmp Primers TaqMan® Array MicroRNA Cards Megaplex™ RT Primers

• Pre-configured arrays correspond to Megaplex Primer Pools A and B

• Up to 381 miRNA targets per array

• Comprehensive coverage of the Sanger miRBase v14 or v10 (for human or rodent, respectively)

Pools of RT primers from TaqMan® MicroRNA Assays

Pools of forward and reverse PCR primers from TaqMan® MicroRNA Assays

Optional

TaqMan® MicroRNA Assays

• Up to 381 multiplexed forward and reverse assay primer pairs

• Preamplification of cDNA prior to real-time PCR when working with small samples (<350 ng)

• Single tube RT reaction with up to 381 Megaplex RT Primers

• Generate cDNA in single reactions for all assays on a TaqMan® Array MicroRNA Card

• Each assay contains a target-specific RT primer and a primer/probe set for real-time PCR

• Assay availability is regularly updated with new human, mouse, and rat sequences in the miRBase database

TaqMan® MicroRNA Assay

1 3 4 5 6 72 8 1 3 4 5 6 72 8

TaqMan® Array MicroRNA CardsCARD A CARD B

F

R

F Q

F

R

F Q

F

R

F Q

F

R

F Q

F

R

F Q

miRNA-specificstem-loop RT primer

PCR primer pair& TaqMan® probe

Megaplex™ RT PrimersPOOL A

Megaplex™ RT PrimersPOOL B

Megaplex™™

PreAmp PrimersPOOL A

Megaplex™ PreAmp PrimersPOOL B

F

R

F

R

F

R

F

R

F

R

F

R

F

R

F

R

F

R

F

R

Megaplex™ Primer Pools

Figure 13.10. MicroRNA detection using Megaplex™ Primer Pools and TaqMan® Array MicroRNA Cards. Syntheticartificialtargets(10pM)foreachassayrepresentedontheAandBTaqMan®ArrayMicroRNACardswerespikedintoacomplextotalRNAbackground(10ng/μL).Themixturewasthenseriallydilutedacrossarangeof6or4logs,anddetectionoftheartificialtargetswastestedusingtheMegaplex™primerpoolsworkflowwithandwithoutthepreamplificationstep.Inaddition,no-templatecontrol(NTC)reactionswereperformedtoconfirmassayspecificity.Subsequently,real-timePCRquantitationisperformedusingcorrespondingTaqMan®ArrayHumanorRodentMicroRNACards,onanAppliedBiosystems®7900HTFastReal-TimePCRSystem.

Concentration of template Concentration of template

Detection Rate of Megaplex™ Human A Pool Content Detection Rate of Megaplex™ Human B Pool Content

Det

ectio

n %

RT PreAmp

0NTC 0.1 fM 1 fM 10 fM 100 fM 1 pM 10 pM 100 pM

20

40

60

80

100RT PreAmp

0NTC 0.1 fM 1 fM 10 fM 100 fM 1 pM 10 pM 100 pM

20

40

60

80

100

13Overview of m

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TaqMan® Array MicroRNA CardsIdeal forhumanor rodentprofiling,TaqMan®ArrayMicroRNACardsprovideall theadvantagesofTaqMan®MicroRNAAssays inaconvenient,

preconfiguredmicrofluidiccard.ThecontentofeachcardismatchedtotherespectiveMegaplex™PrimerPoolsandcontainsupto381unique

TaqMan®MicroRNAAssays,reducingsetuptimeandexperimentalvariability.FollowingreversetranscriptionofmiRNAtargetsusingMegaplex™

RTPrimers,andoptionalpreamplificationwithMegaplex™PreAmpPrimers,TaqMan®UniversalMasterMixIIissimplycombinedwitheachreac-

tionandpipettedintoeachoftheTaqMan®Arraycardseightsampleloadingports.Thissimplifiessamplehandlingandhelpsincreasesample

throughput.AsetoftwoTaqMan®MicroRNAArraysforeachspeciesprovidesassaystocoverSangermiRBasev14orv10(forhumanorrodent,

respectively)databasecontent.WhenusedinconjunctionwithMegaplex™RTPrimersandoptionalMegaplex™PreAmpPrimers,acomprehen-

sivedatasetcanbegeneratedinaslittleasfivehours.

Integromics StatMiner® SoftwareRealTimeStatMiner®SoftwarefromIntegromicsisapowerful,easy-to-usebioinformaticstoolforqualitycontrolanddifferentialexpressionanaly-

sisofreal-timePCRdata.Withjustasingleclick,thesoftwareimportsrawdataorCTvaluesfromanyAppliedBiosystems®Real-TimePCRSystem,

andthenguidesyouthougheverystepofanalysistoenableaccurateresultsinminutes.Thesoftwareisidealformedium-andhigh-throughput

PCRexperimentsandiscompatiblewithTaqMan®ArrayCards,andTaqMan®Assaysinplateformat.

TaqMan® MicroRNA Reverse Transcription (RT) KitTheTaqMan®MicroRNARTKitprovidesallthenecessarycomponentsforoptimalTaqMan®MicroRNAAssayperformance.Componentsofthiskit

areusedwiththesingleRTprimerprovidedwitheachindividualTaqMan®MicroRNAAssay,orwithMegaplex™RTPrimers,toconvertmiRNAto

cDNApriortoreal-timePCRquantitation.

Detect and quantify specific microRNAs

DetailedstudiesonspecificmiRNAsareoftenconductedtovalidatepreviousresultsortolearnmoreaboutthesemiRNAs.Throughtheuseofnovel

adaptationsinassaydesign,AppliedBiosystemsbringsthebenefitsofTaqMan®Assaysandquantitativereal-timePCR—unparalleledsensitivity,specific-

ity,anddynamicrange—tomiRNAdetectionandquantitation.TaqMan®MicroRNAAssaysincorporateatarget-specificstem-loop,reversetranscription

primer.ThisinnovativedesignaddressesafundamentalprobleminmiRNAquantitation:theshortlengthofmaturemiRNAs(~22nt).Thestem-loop

structureprovidesspecificityforonlythematuremiRNAtargetandformsanRTprimer/maturemiRNAchimerathatextendsthe3’endofthemiRNA.

Theresulting,longerRTproductpresentsatemplateamenabletostandardTaqMan®real-timePCRassay.Tofacilitateaccurateresults,everyTaqMan®

MicroRNAAssay is functionallyvalidatedunder laboratoryconditions.AppliedBiosystemsoffersacomprehensivecollectionofTaqMan®MicroRNA

Assayscoveringabroadcollectionofspecies,includingbutnotlimitedtohuman,mouse,rat,Arabidopsis,C. elegans,andDrosophila.Inaddition,new

assaysareaddedonaregularbasisinaccordancewithupdatestotheSangermiRBaseRegistry.

Targeted microRNA quantitation workflowFeaturesoftheworkflow(Figure13.11):

→ Ideal for quantitating individual miRNAs from a broad assortment of species, including human, mouse, rat, Drosophila, C. elegans, and

Arabidopsis

→ EachkitincludestheTaqMan®AssayandreversetranscriptionprimerspecificforthematuremiRNAtargetofinterest

→ Assaysarecontinuouslyaddedforhuman,mouse,andratspeciestostayalignedwiththeSangermiRBaseRegistry

How it worksTaqMan®MicroRNAAssaysemployan innovative target-specificstem-loop reverse transcriptionprimer toaddress thechallengeof theshort

lengthofmaturemiRNA.Theprimerextendsthe3’endofthetargettoproduceatemplatethatcanbeusedinstandardTaqMan®Assay-based

real-timePCR(Figure13.12).Also,thestem-loopstructureinthetailoftheprimerconfersakeyadvantagetotheseassays:specificdetectionof

themature,biologicallyactivemiRNA.

Individual TaqMan® MicroRNA AssaysTwo-step TaqMan® MicroRNA Assays, containing the TaqMan® Assay and reverse transcription primer specific for the target of interest, are

designedforindividualmiRNAtargetsfoundinhuman,mouse,rat,Drosophila,C. elegans,andArabidopsisspecies.AppliedBiosystemsiscontinu-

ouslyincreasingthenumberofassaysforthesespeciestoremainalignedwiththeSangermiRBaseRegistry.DuetothehighdegreeofmiRNA

conservationbetweenspecies,coverageisobservedtoextendwellbeyondthesecorespecies.

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Set Up Singleplex TaqMan® MicroRNA

Assay Reaction

Perform Real-Time PCRAmplification

Analyze DataReverse

Transcribe RNA

• Ambion® mirVana™ miRNA Isolation Kit

• RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE

• TaqMan® MicroRNA Cells-to-CT™ Kit

• TaqMan® MicroRNA Reverse Transcription Kit

• TaqMan® MicroRNA Assays

• TaqMan® Universal Master Mix II

• TaqMan® MicroRNA Assays

• Applied Biosystems® Real-Time PCR System

• Instrument Software (included)

• RQ Manager Enterprise Software (optional)

• RealTime StatMiner® Software (optional)

Prepare Sample

30 min 65 min 25 min 90 min

Stem-loop primer

microRNAmicroRNA

Figure 13.11. Targeted microRNA quantitation workflow.

Q F

Reverse Transcription

Real-Time PCR

Stem-loop RT primer

Target Sequence

Forward Primer

TaqMan® ProbeReverse Primer

Figure 13.12. TaqMan® MicroRNA Assay approach. Asimpletwo-stepprocessbringstheadvantagesofreal-timePCRtomiRNAresearch.

TaqMan® MicroRNA Assay Endogenous ControlsThisselectionofendogenouscontrolassaysforhuman,mouse,rat,Arabidopsis,C. elegans,andDrosophilasimplifiesdatanormalization.Designed

againstcarefullyselectedsmallnon-codingRNAsthatareunrelatedtomiRNAs,thesecontrolsareexpressedatconsistentlevelsacrossawide

varietyofcelltypes,tissues,andexperimentalconditions.

TaqMan® MicroRNA Reverse Transcription (RT) KitTheTaqMan®MicroRNARTKitprovidesallthenecessarycomponentsforoptimalTaqMan®MicroRNAAssayperformance.Componentsofthiskit

areusedwiththesingleRTprimerprovidedwitheachindividualTaqMan®MicroRNAAssay,orwithMegaplex™RTPrimers,toconvertmiRNAto

cDNApriortoreal-timePCRquantitation.

Custom TaqMan® Small RNA Assays (Early Access)ThenoveladaptationsinTaqMan®AssaydesigndevelopedforthestudyofmiRNAsusingTaqMan®MicroRNAAssaysareidealforanalysisofany

smallnucleicacidlessthan200baseslong.WithCustomTaqMan®SmallRNAAssays,thebenefitsoftheseassaysareavailableforanysmallRNA,

fromanyspecies.ThisincludesnewlydiscoveredmiRNAsthatarenotyetintheregistryandotherclassesofsmallRNAs,suchaspiwi-interacting

RNA(piRNA),smallnuclearRNA(snRNA),andsmallnucleolarRNA(snoRNA).

13Overview of m

iRNAs



Figure 13.14. TaqMan® MicroRNA Assays provide wide dynamic range. ThiswidedynamicrangeenablesmiRNAtargetsthatvaryinabundancefromafewcopiestomillionsofcopiestobeaccuratelyquantitatedinthesameexperiment—animportantfactorgiventhewiderangeofmiRNAconcentrationswithinandacrossdifferentcells,tissuetypes,anddiseasestates.ToillustratethedynamicrangeandsensitivityofTaqMan®MicroRNAAssays,asyntheticlin-4miRNAwasseriallydilutedandamplifiedusingthelin-4TaqMan®MicroRNAAssay.(A)Amplificationplotofsyntheticlin-4miRNAoversevenordersofmagnitude.SyntheticRNAinputrangedfrom1.3x10–3fM(equivalentto7copiesperreaction)to1.3x104fM(equivalentto7x107copiesperreaction)inPCR;(B)Standardcurveofsyntheticlin-4miRNAamplification.

Ampli�cation Plot

Cycle Quantity

Standard Curve

1.0

39

37

35

33

31

29

27

25

23

21

19

17

15

13

11

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0 5 10 15 20 25 30 35 40 10 100 1,000 104 105 106 107 108 109

Slope = –3.36R2 = 0.999

7copies

70Mcopies

miRNA Assays

Mature miRNAs Precursors

Perfectly matched CT

Mismatched CT ∆CT (mismatch vs. match)

CT ∆CT (Precursor vs. mature)

let-7aLooped 16.5 33.1 16.6 29.5 13.0

Linear 23.6 38.3 14.7 30.4 6.8

miRNA

Stem-loopRT primer

miRNA

LinearRT primer

Figure 13.13. The stem-loop primer strategy for reverse transcription in TaqMan® MicroRNA Assays confers specificity for biologically active mature microRNA. Anoff-the-shelfTaqMan®MicroRNAAssayforlet-7a,containingastem-loopRTprimer,wascomparedwithacomparable-sequencelinearRTprimer/PCRprimer/TaqMan®probeset.Next,1.5x108copiesofsyntheticmiRNAmimickingmaturelet-7a,maturelet-7e(acloselyrelatedmiRNAdifferingatonlytwobasepositions),andthestem-looplet-7aprecursorwereaddedtoRTreactionsprimedwitheitherthestem-loopTaqMan®MicroRNAAssayRTprimerorlinearRTprimerofcomparablesequence.ThecDNAwasthenamplifiedusingreal-timePCR.Thedataindicatethatthestem-loopRTprimerconfersbetterdis-criminationbetweenmatureandprecursormiRNAsandcloselyrelatedtargets.

TaqMan® technology goes small with big benefits for miRNA research→ Highlyspecific—quantitateonlythebiologicallyactivematuremiRNAs,notprecursors—withsingle-basediscriminationofhomologous

familymembers(Figure13.13)

→ Sensitive—requiresonly1–10ngoftotalRNAorequivalenttoconservelimitedsamples

→ Widedynamicrange—upto9logs—detecthighandlowexpressorsinasingleexperiment(Figure13.14)

→ Fast,simple,andscalable—two-stepreal-timeRT-PCRassayquicklyhelpsprovidehigh-qualityresults

→ Customassaysavailable—youspecifythesequence,andAppliedBiosystemswilldesignanassay

Analyze microRNA function

AnalysesofmiRNAfunctionareperformedusingstrategiesthataresimilartothoseusedforprotein-encodinggenes.Transfectingculturedcells

withmiRNAmimicscanhelpidentifygain-of-functionphenotypes;down-regulationorinhibitionexperimentsusingmiRNAinhibitorscanbe

conductedtoidentifyloss-of-functionphenotypes.Thecombinationofup-regulationanddown-regulationcanbeusedtoidentifygenesand

cellularprocessesthatareregulatedbyspecificmiRNAs.FurtherinvestigationofmiRNAfunctionincludesstudiesofmiRNA-mRNAtargetinterac-

tionandtheimpactontargetmRNAlevelsandconcomitantproteinexpression.

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mRNA

5'Cap AAAAAAAAAAAAA3'

5'Cap AAAAAAAAAAAAA3' No translation

Translation

3' 5'

Sequence-specific loading into RISC-like complex

Pre-miR™ Precursor

Complementary binding of specific miRNAs—no RISC-like complex association

Anti-miR™ Inhibitor

Pre-miR™ miRNA Precursors

Anti-miR™ miRNA Inhibitors

3' 5'

3' 5'

3' 5'

3' 5'

3' 5'

Figure 13.15. Activity of Ambion® Pre-miR™ miRNA Precursors and Anti-miR™ miRNA Inhibitors.

Cel

l num

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0

20

40

60

80

100

120

140

160

180

200Negative controlmiR-31 inhibitormiR-24 miR-21

miR-190 miR-218

Figure 13.16. Identification of miRNAs involved in cell proliferation by screening with Anti-miR™ miRNA Inhibitors. HeLacellsweretransfectedwithindividualAnti-miR™miRNAInhibitors.After72hours,cellswerefixed,stainedwithpropidiumiodide,andtreatedwithanantibodytodetectβ-actin.CellnumberwasanalyzedusinganAcumenExplorer™(TTPLabTech)platereader.ThehorizontalshadedarearepresentsthenormalrangeofcellnumberforthistypeaftertreatmentwithAnti-miR™NegativeControl#1.Therightinsetshowsthemorphologyofthesecontrolcells.TheleftinsetshowsthemorphologyofcellstransfectedwithanAnti-miR™InhibitortomiR-31.Artificialup-regulationofmiR-31resultedinelongatedcellsandreducedcellnumbers.

Ambion® Pre-miR™ miRNA Precursors and Anti-miR™ mimics and inhibitors and the means to deliver them to cellsAmbion®Pre-miR™miRNAPrecursorsandAnti-miR™miRNAInhibitorsaredesignedtomimicorinhibitspecificmiRNAsforgain-of-functionorloss-of-function

studies,respectively(Figure13.15).BothcanbeintroducedintocellsusingtransfectionorelectroporationparameterssimilartothoseusedforsiRNAs.

Pre-miR™miRNAPrecursorsaresmall,chemically-modifieddouble-strandedRNAmoleculesthataresimilarto,butnotidenticalto,siRNAs,

andaredesignedtomimicendogenousmaturemiRNAs.Thechemicalmodificationsensurethatthecorrectstrand,representingthedesired

maturemiRNA,istakenupintotheRNA-inducedsilencingcomplex(RISC)-likeresponsibleformiRNAactivity.Thankstotheirsmallsize,theyare

easiertotransfectthanvectors,andcanbedeliveredusingconditionssimilartothoseusedforsiRNAsbytransfectionorelectroporation.Incon-

trasttomiRNAexpressionvectors,thesesyntheticmoleculescanbeusedindoseresponsestudies.

Ambion®Anti-miR™miRNA Inhibitorsarechemicallymodified, single-strandednucleicacidsdesignedtospecificallybindtoand inhibit

endogenousmiRNAs.WhentestedusingindividualreporterconstructscontainingtheappropriatemiRNAbindingsite,theseinhibitorsinduced,

onaverage,anapproximately4-foldincreaseintheexpressionofthereporterrelativetocellscotransfectedwithanegativecontrolAnti-miR™

miRNAinhibitor,indicatingtheirstronginhibitoryproperties.

Thereisasuiteofproductsbasedonthistechnologythatincludespositiveandnegativecontrols,aswellasPrecursorandInhibitorLibrariesavail-

ableaspremadeorcustomsets.Figure13.16showsanexperimentinwhichalibraryofAnti-miR™miRNAInhibitorswasscreenedtoidentifymiRNAsthat

areinvolvedincellproliferation.NotethatAmbion®Pre-miR™miRNAPrecursorsandAnti-miR™miRNAInhibitorskeeppacewithnewadditionstothe

miRBasedatabase,andforsequencesthatarenotpublished,customer-definedmimicsandinhibitorsareavailable.

OrderPre-miR™andAnti-miR™productsatwww.invitrogen.com/ordermirna.

13Overview of m

iRNAs



Pre-miR™ miRNA Precursor LibrariesThePre-miR™miRNAPrecursorLibrary-HumanV3consistsof470miRNAmimicscorrespondingto470humanmaturemiRNAscatalogedinver-

sion9.2ofthemiRBaseSequenceDatabase.ThiscollectionofmiRNAmimicsenablesrapidstudyofmiRNAfunction(Figure3)andcanbeused

tosimplifyidentificationofmiRNAsthatinteractwithaparticulartargettranscript.EachPre-miR™miRNAPrecursorisinaquantityof0.25nmol,

driedinindividualwellsofa96-wellplate.Thisamountissufficientfor250transfectionswhenusedat10nMin96-wellplates.

ThePre-miR™miRNAPrecursorLibrary–MouseV3consistsof379miRNAmimicscorrespondingto379mousematuremiRNAscataloged

inversion9.2ofthemiRBaseSequenceDatabase.ThiscollectionofmiRNAmimicsenablesrapidstudyofmiRNAfunctionandcanbeusedto

simplifyidentificationofmiRNAsthatinteractwithaparticulartargettranscript.EachPre-miR™miRNAPrecursorissupplieddriedat0.25nmolin

individualwellsofa96-wellplate.Thisamountissufficientfor250transfectionswhenusedat10nMin96-wellplates.

Custom Pre-miR™ miRNA Precursor Libraries, which include 50 or more Pre-miR™ miRNA Precursors of your choice in the amount you

specify,arealsoavailable.Contactusatepiscientist@invitrogen.comforadditionaldetails.

Anti-miR™ miRNA Inhibitor LibrariesTheAnti-miR™miRNAInhibitorLibrary-HumanV2 includes327miRNAinhibitors (0.25nmoleach)thatcorrespondto327miRNAs inmiRBase

SequenceDatabaseversion8.0.

TheAnti-miR™miRNAInhibitorLibrary–MouseV3consistsof379miRNAinhibitorscorrespondingto379mousematuremiRNAscataloged

inversion9.2ofthemiRBaseSequenceDatabase.ThiscollectionofmiRNAinhibitorsenablesrapidstudyofmiRNAfunctionandcanbeusedto

simplifyidentificationofmiRNAsthatinteractwithaparticulartargettranscript.EachAnti-miR™miRNAInhibitorissupplieddriedat0.25nmolin

individualwellsofa96-wellplate.Thisamountissufficientfor50transfectionswhenusedat50nMin96-wellplates.

CustomAnti-miR™miRNAInhibitorLibraries,whichinclude50ormoreAnti-miR™miRNAInhibitorsofyourchoiceintheamountyouspecify,

arealsoavailable.Contactusatepiscientist@invitrogen.comforadditionaldetails.

Orderatwww.invitrogen.com/epi.

Reporter vector and combined chemiluminescent assay systemsTo evaluate the interaction between miRNAs and their target sites, the Ambion® pMIR-REPORT™ miRNA Expression Reporter Vector System

provides a simple solution. This validated reporter gene system contains two mammalian expression vectors, pMIR-REPORT™ Luciferase and

pMIR-REPORT™ β-Galactosidase Control Vector. pMIR-REPORT™ Luciferase features the firefly luciferase reporter gene under the control of a

CMVpromoterandacloning region formiRNAtargetsequencesora3’UTRwithoneormoreputativemiRNAbindingsitesdownstreamof

the luciferasecodingsequence.Aftercloning,pMIR-REPORT™Luciferasecanbecotransfected intomammaliancellswith thepMIR-REPORT™

β-GalactosidaseControlVectortoevaluatetheeffectsofendogenousmiRNAexpressiononthetarget.

Inaddition,thepMIR-REPORT™Systemcanbeusedtomonitordown-regulationorup-regulationofreportergeneexpressionaftertrans-

fectionwithPre-miR™miRNAPrecursorsorAnti-miR™miRNAInhibitors,respectively.WithaPre-miR™miRNAPrecursorLibrary,pMIR-REPORT™

LuciferaseisanidealscreeningtooltostudymiRNA-mediatedregulationofatargetgene.

Theresultingluciferaseandβ-galactosidaseactivitycanbemeasuredusingtheTropix®Dual-Light®Luciferaseandβ-GalactosidaseReporter

GeneAssaySystem.Withthisinnovativesystem,bothassaysareperformedinlessthanonehourusingasingleextractforgreaterconvenience

andprecision.Moreover,thewidedynamicrangeofthisdualassayenablesaccuratemeasurementofluciferaseandβ-galactosidaseconcentra-

tionsoversevenordersofmagnitude,with100-to1,000-foldgreatersensitvitythancolorimetricandfluorescentassaysforβ-galactosidase.

Protein expression analysismiRNAfunctionalstudiesmayrequiresimultaneousanalysesofRNAandproteinexpression.TheWestern-SuperStar™ImmunodetectionSystem

isahighlysensitivechemiluminescentimmunodetectionsystem,providinganidealsolutionformeasuringtheeffectofmiRNAfunctiononthe

expressionofspecificproteins.

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TaqMan® Gene Expression AssaysAnumberofmiRNAshavebeenreportedtodirectlyaffecttargetmRNAlevels.Othersarethoughttotargettheexpressionoftranscriptionfactors,

indirectlyaffectingtheexpressionlevelsofmanygenes.Eitherway,TaqMan®GeneExpressionAssayscanbeusedwithmiRNAgain-of-function

andloss-of-functionexperimentstoquantitateeffectsontargetmRNAexpression.

Applied Biosystems offers more than 1,000,000 TaqMan® Gene Expression Assays for 19 species, the most comprehensive set of pre-

designed,real-timePCRassaysavailable.AllTaqMan®GeneExpressionAssaysaredesignedusingourvalidatedbioinformaticspipeline,andrun

withthesamePCRprotocol,eliminatingtheneedforprimerdesignorPCRoptimization.

MicroRNA functional analysis workflowFeaturesoftheworkflow(Figure13.17):

→ Gain-andloss-of-functionphenotypesidentifygenesandprocessesregulatedbymiRNAs

→ Westernblottingand/orreal-timeRT-PCRareusedtovalidatemiRNAtargets

→ Targetsiteinteractionandimpactonproteinexpressioncanbeevaluatedusingreportergeneandimmunodetectionsystems

How it worksAmbion®Pre-miR™miRNAPrecursorsandAnti-miR™miRNAInhibitorsaredesignedtomimicorinhibitspecificmiRNAsforartificialup-regulation

and down-regulation of target mRNA translation, respectively. miRNA targets are validated by quantitating target protein and/or messenger

RNAlevelsinreponsetomiRNAup-regulationordown-regulation.Westernblotanalysisisusedtoinvestigatetheimpactonproteinexpression.

Assess target mRNA levels(optional)

assess target protein levelsor other biological effect

Transfect cells

• Ambion® Anti-miR™ miRNA Inhibitors• Ambion® Pre-miR™ miRNA Precursors

• Ambion® Pre-miR™ miRNA Starter Kit• Lipofectamine® RNAiMAX

• TaqMan® Gene Expression Cells-to-CT™ Kit• TaqMan® Gene Expression Assays• Applied Biosystems Real-Time PCR Systems

• Western-SuperStar™ Immunodetection System• Ambion® pMIR-REPORT™ miRNA Expression Reporter Vector System• Dual-Light® Combined Reporter Gene Assay System

Select miRNA inhibitoror mimics and controls

Pre-miR™ miRNA Precursort

Anti-miR™ miRNA Inhibitor

3' 5'3' 5'

Figure 13.17. MicroRNA functional analysis workflow.

13Overview of m

iRNAs



Sample preparation tailored for microRNA experiments

MostRNAisolationkitsweredevelopedtorecovermessengerRNA,andeliminatesmallermoleculessuchasmiRNAs.Invitrogenoffersarangeof

productsthatweredesignedforoptimalrecoveryofmiRNAandothersmallRNAsfromawidevarietyofsampletypes(Figure13.18).Theselection

guide(Table13.1)isdesignedtohelpresearchersdecidewhichproductbestfitstheirneeds.Thefollowingpagesprovidemoredetailedinforma-

tiononthesamplepreparationproductsmentionedintheworkflowsformiRNAdiscovery,profiling,targetedanalysis,andfunctionalanalysis

MicroRNA Sample Preparation KitsInvitrogenoffersavarietyofkitsfortheisolationandanalysisofmiRNAandothersmallRNAs.EachkitisidealforuseinmiRNAanalysisbecause

theyareoptimizedtoenable:

→ QuantitativerecoveryofsmallRNA(<200nt)

→ MaintenanceofrepresentativeamountsofsmallRNA(eliminatingexperimentalbias)

Ambion® mirVana™ miRNA Isolation KitSamplesarelysedinadenaturinglysissolutionthatbothstabilizesRNAandinactivatesRNases.Thelysateisthenextractedwithacid-phenol/

chloroform,yieldingasemipureRNAsample.TheRNAisfurtherpurifiedoveraglass-fiberfiltertoyieldeithertotalRNAorasizefractionenriched

inmiRNAs.ThekitreagentsarespecificallyformulatedformiRNAretentiontoavoidthelossofsmallRNAsthatistypicallyseenwithstandard

glass-fiberfiltermethods.Thisquickandeasyprocedureiscompatiblewithvirtuallyallcellandtissuetypes,andcanbeusedforefficientisolation

ofsmallRNA-containingtotalRNA,orforenrichmentofthesmallRNAfraction(<200nt),toincreasesensitivityindownstreamanalyses.

MicroRNAs Are you isolating RNA from blood?

RecoverAll™

Kit

Yes

No Are you isolating RNA from FFPE tissue?

Yes

No

Blood types

LeukoLOCK™

Kit

Human

Mouse RiboPure™

Blood Kit

Mouse

Would you like to isolate

both RNA & protein?

mirVana™

PARIS™ KitYes

No Isolated RNA needs to...

Contain microRNA

Be enriched for microRNA

mirVana™

miRNA Isolation

Kit

>10 mg tissue>105 cells

<10 mg tissue<105 cells

≤105 cells

How much starting material?

mirVana™

miRNA Isolation

Kit

Would you like to skip the

RNA puri�cation step?

Yes

No

TaqMan®

MicroRNACells-to-CT™

Kit

RNAqueous®

Micro Kit

RNAqueous®

Micro Kit

Figure 13.18. MicroRNA sample preparation selection guide.

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MicroRNAs Are you isolating RNA from blood?

RecoverAll™

Kit

Yes

No Are you isolating RNA from FFPE tissue?

Yes

No

Blood types

LeukoLOCK™

Kit

Human

Mouse RiboPure™

Blood Kit

Mouse

Would you like to isolate

both RNA & protein?

mirVana™

PARIS™ KitYes

No Isolated RNA needs to...

Contain microRNA

Be enriched for microRNA

mirVana™

miRNA Isolation

Kit

>10 mg tissue>105 cells

<10 mg tissue<105 cells

≤105 cells

How much starting material?

mirVana™

miRNA Isolation

Kit

Would you like to skip the

RNA puri�cation step?

Yes

No

TaqMan®

MicroRNACells-to-CT™

Kit

RNAqueous®

Micro Kit

RNAqueous®

Micro Kit

Ambion® RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE TissuesTheRecoverAll™Kitprocedurerequiresabout45minutesofhands-ontimeandcaneasilybecompletedinlessthan1.5hourswhenisolating

RNA.FFPEsamplesaredeparaffinizedusingaseriesofxyleneandethanolwashesandarethensubjectedtoarigorousproteasedigestionwith

anincubationtimetailoredforrecoveryofeitherRNAorDNA.Nucleicacidsarepurifiedusingarapidglass-filtermethodthatincludesanon-filter

nucleasetreatment,andarefinallyelutedintoeitherwaterorthelow-saltbufferprovided.ThedegreeofRNAfragmentationthathasalready

occurredinFFPEtissuescannotbereversed.However,theproteasedigestionconditionsoftheRecoverAll™kitaredesignedtoreleaseamaximal

amountofRNAfragmentsofallsizes,includingmiRNA,inarelativelyshortamountoftime.ThisRNAcanbereadilyanalyzedbyreal-timeRT-PCR

andgeneratesprofilesequivalenttothoseseeninRNAisolatedfromfreshorflash-frozensamples.

TaqMan® MicroRNA Cells-to-Ct™ KitStartwith10–100,000culturedcellspersample,either inmultiwellplatesor individualtubes,andbereadyforRT-PCRin10minutes.Cellsare

washedinPBSandlysedfor8minutesatroomtemperature;DNasetreatmentcanbeperformedconcurrently.LysisisterminatedbyaddingStop

Solutionandincubatingfortwoadditionalminutesatroomtemperature.Becausesamplescanbeprocesseddirectly incultureplates(96-or

384-well),samplehandlingisreduced,andtheriskofsamplelossortransfererrorisminimized.Noheating,washing,orcentrifugationisrequired;

theTaqMan®MicroRNACells-to-Ct™Kitgreatlyreducesatraditionallytime-consuming,labor-intensiveprocesstojust10minutes.Alsoincluded

inthekitareTaqMan®MicroRNAReverseTranscriptionReagentsandTaqMan®UniversalPCRMasterMixtocompletethegoldstandardTaqMan®

miRNAprofilingworkflow.

TaqMan® Gene Expression Cells-to-Ct™ KitSimilartotheCells-to-Ct™KitformicroRNA,theTaqMan®GeneExpressionCells-to-Ct™Kitenablesreal-timeRT-PCRdirectlyinculturedcelllysates

withoutisolatingRNA.Thiskit,however,includesreversetranscriptionandreal-timePCRreagentsthatareoptimizedforquantitationofmRNA

expression.ItisidealforvalidationstudiesthatanalyzetheeffectsofmiRNAontheirmRNAtargets.

Figure 13.18. MicroRNA sample preparation selection guide.

13Overview of m

iRNAs



1. MattickJS(2001)EMBO Rep2:986–991.

2. AmbrosV(2004)Nature431:350–355.

3. BentwichIetal.(2005)Nat Genet37:766–770.

4. OkazakiYetal.(2002)Nature420:563–573.

5. ImanishiTetal.(2004)PLoS Biol2:e162.

6. RhoadesMWetal.(2002)Cell110:513–520.

7. ChenX(2005)FEBS Letters579:5923–5931.

8. ElbashirSetal.(2001)Genes Dev15:188–200.

9. LimLPetal.(2003)Science299:1540.

10. KimVN,NamJW(2006)Trends Genet22:165–173.

11. BartelDP(2004)Cell116:281–297.

12. SoiferHS,RossiJJ,SaetromP(2007)Mol Ther15:2070–2079.

13. Griffiths-JonesS,SainiHK,vanDongenSetal.(2008)Nucleic Acids Res36:D154–D158.

Table 13.1. MicroRNA sample preparation kits: quantitative recovery of small RNAs from a variety of sample types.

TaqMan® MicroRNA Cells-to-Ct™ Kit

mirVana™ miRNA Isolation Kit mirVana™ PARIS™ Kit

RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE Tissues

MagMAX™-96 for Microarrays Total RNA Isolation Kit

Technology Cells-to-Ct™technology,withoptionalDNasetreat-mentforpreparationofculturedcelllysatesthatcanbeusedinreal-timeRT-PCRwithoutRNAisolation

Acid-phenolandrapid,enhancedglass-fiberfilterpurification

CellDisruptionBuffercombinedwithacid-phenol/chloroformextractionandglass-fiberfilterpurification

Deparaffinization,proteasedigestion,andglass-fiberfilterpurification

CelllysisusingTRIREAGENT®andmagneticbeadpurification

Sample input amounts

10to105culturedcells 103–107culturedcellsor0.5–250mgtissue

100–107culturedcellsorupto100 mgtissue

Uptofour20μmFFPEsections Upto5x106culturedcellsorupto100mgtissue

Features • GofromcellsinculturetoRT-PCR,typicallyin10minatroomtemperature

• Simpleprocedurewithnosampletransfers,nocentrifugation,andnovacuummanifoldneeded

• Forreal-timeRT-PCR• Superiorresultswhen

usedwithTaqMan®MicroRNAAssaysandArrays

• Fast,easyisolationofsmallRNAfromculturedcellsandmosttissues(includingtissueswithhighlevelsofribonucleases)

• IdealformiRNAprofilingexperi-mentsandothergeneexpressionapplications

• Simple,30minuteprocedure

• Proteincanalsoberecovered

• OptionalsmallRNAenrichmentprocedure

• Idealforcorrelat-ingmRNA,miRNA,and/orsiRNAwithproteinlevels

• Isolatetotalnucleicacids,includingDNAandmicroRNAs,fromFFPEsamples

• NoovernightproteinaseKdigestionrequired—deparaf-finizeinthemorningandper-formqRT-PCRintheafternoon

• Routinelyobtainyieldsof>50%thatofunfixedtissuefromthesamesamplesource

• Forreal-timeRT-PCRandPCR,mutationscreening,andmicroarrayanalyses

• Highlyconsistentresultsfromexperi-menttoexperiment

• StreamlinedRNApurification

• Requireslesshands-ontimethancompetitorkits

• Walkaway—integratewithestablishedroboticplatforms

• ModifiedprotocoltorecoversmallRNAs

Kit sizes and part numbers

100lysisrxns/500PCRsP/N4391848400lysisrxns/2,000PCRsP/N4391996

Upto40purificationsP/NAM1560

Upto40purificationsP/NAM1556

40purificationsP/NAM1975

96purificationsP/NAM1839

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Chapter 14Optimized miRNA profiling

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CHAPTER 14

Optimized miRNA profiling

NCode™ platform—for optimized miRNA profiling

miRNAexpressionprofilingcameintoprominenceinpartbecauseoftheexpectationthatahighlyexpressedmiRNAforagiventissueorcelltype

(ordevelopmentalstage)islikelytoplayaregulatoryrole.SincethefirstpublishedarticletoreportonmiRNAprofilingusinganoligonucleotide

microarray[1],microarrayanalysishasbecomethepreferredtoolforprofilingmiRNAexpressionpatternstogaininsightintotheirrelevancein

developmentanddisease.TheNCode™platformisoptimizedtoinvestigateglobalmiRNAexpressionpatterns.Fromsamplepreparationtoarray

analysistoqRT-PCRvalidation,theNCode™platformprovidesoptimizedreagentsforeverystepofthemiRNAprofilingworkflow(Figure14.1).

Total RNA isolationTRIzol® Reagent

miRNA labelingNCode™ miRNA Ampli�cation System

NCode™ Rapid miRNA Labeling System

miRNA data analysisNCode™ Pro�ler software

miRNA qRT-PCRNCode™ SYBR® Green and SYBR® GreenER™ qRT-PCR Kits

NCode™ miRNA First-Strand cDNA Synthesis Kit

Preprinted miRNA microarray

NCode™ Human miRNA Microarray V3NCode™ Multi-Species miRNA Microarray V2

In-house printed miRNA microarray

NCode™ Human miRNA Microarray Probe Set V3NCode™ Multi-Species miRNA Microarray Probe Set V2

miRNA microarray controlsNCode™ Multi-Species miRNA

Microarray Control V2

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Functional toolsAmbion® Pre-miR™ miRNA precursorsAmbion® Anti-miR™ miRNA inhibitors

Figure 14.1. NCode™ platform workflow. Duringsamplepreparation,totalRNAisisolatedandquantified.AmplificationofthesmallRNAmoleculesmaybenecessary when there is insufficient starting material. Next, a direct labeling method polyadenylates the enriched small RNA molecules and ligates afluorescentlyconjugated,branchedDNAstructuretothetailedRNA.TaggedandtailedmiRNAsaresubsequentlyhybridizedtothearray.BoundmiRNAsaredetectedbythehybridizationofbranchedDNAstructurescontainingdyemolecules.ThemiRNAexpressionprofileiscalculatedfromrelativesignalintensitydetectedbyamicroarrayscannerforeachspotonthearray.DatavalidationisaccomplishedusingqRT-PCR.

14Optim

ized miRNA profiling



Efficient isolation of total RNA (including miRNA) using TRIzol® Reagent

Isolatinghigh-qualitytotalRNAfromyoursampleisastepcrucialtothesuccessofyourresearch.TRIzol®ReagentpurifiesallRNAs,includingthose

inthe10–200nucleotiderange,andisthusrecommendedforisolatingtotalRNAforstudyingmiRNAs.Extractionproceduresinvolveready-to-

usemonophasicsolutionsofphenolandguanidineisothiocyanate,andarebaseduponimprovementstothesingle-stepRNAisolationmethod

developedbyChomczynskiandSacchi[2].UsingTRIzol®Reagentenables:

→ MoreeffectivepurificationoftotalRNAthancolumn-basedpurification,withoutdepletionofsmallRNAs

→ Asimple,flexibleprotocol,completedinlessthan1hr

→ High-purityRNAfromawiderangeofanimaltissuesandcells

Reliable recovery of total RNA (including miRNA) from FFPE samples with the RecoverAll™ Total Nucleic Acid Isolation Kit

→ Optimizedforisolationoftotalnucleicacids,includingmicroRNAs,fromFFPEtissue

→ NoovernightproteinaseKdigestionrequired—deparaffinizeinthemorningandperformqRT-PCRintheafternoon

→ Obtaintypicalyieldsof>50%thatofunfixedtissuefromthesamesamplesource

→ RecoverednucleicacidsaresuitableforqRT-PCR,qPCR,mutationscreening,andmicroarrayanalyses

TheRecoverAll™TotalNucleicAcidIsolationKitisdesignedtoextracttotalnucleicacidfromformalinorparaformalin-fixed,paraffin-embedded

(FFPE)tissues(Figure14.2).Uptofour20µmsections,orupto35mgofunsectionedcoresamples,canbeprocessedperreaction.

Theabilitytoisolatenucleicacidfromarchivedtissuesamplesthatissuitableformolecularanalysisenablesretrospectivestudiesofdis-

easedtissueatboththegenomicandgeneexpressionlevel.Whilestandardpreservationtechniquesthatemployformalinareidealformaintain-

ingtissuestructureandpreventingdecomposition,formalinpreservationcreatesprotein–proteinandprotein–nucleicacidcrosslinksthatmakeit

difficulttoperformmolecularanalyses.Inaddition,RNA(andtosomeextentDNA)isoftenfragmentedandchemicallymodifiedtosuchadegree

thatitisincompatiblewithmanymolecularanalysistechniques.

ThedegreeofRNAfragmentationthathasalreadyoccurredinFFPEtissuescannotbereversed.However,theproteasedigestionconditions

oftheRecoverAll™Kitaredesignedtoreleaseamaximalamount(Figure14.3)ofRNAfragmentsofallsizes,includingmicroRNA,inarelatively

shortamountoftime.

TheRecoverAll™TotalNucleicAcidIsolationKitprocedurerequiresabout45minutesofhands-ontimeandcaneasilybecompletedinless

than1daywhenisolatingRNA.FFPEsamplesaredeparaffinizedusingaseriesofxyleneandethanolwashes.Next,theyaresubjectedtoarigorous

proteasedigestionwithanincubationtimecompatibleforrecoveryofeitherRNAorDNA.Thenucleicacidsarepurifiedusingarapidglass-filter

methodologythatincludesanon-filternucleasetreatmentandareelutedintoeitherwaterorthelow-saltbufferprovided.

Therecoverednucleicacidsaresuitablefordownstreamapplicationssuchasmicroarrayanalyses,qRT-PCR(seeFigure14.4),andmutation

screening.However,asisthecasewithallFFPEtissue,samplefixationandstoragetypicallycausenucleicacidfragmentationandmodification.

Therefore,downstreamapplications,suchasmicroarrayanalysis,whichrequiremorepristineRNAthandoesqRT-PCR,mayrequiremodification

forbestresults.AlthoughDNAtendsnottofragmentaseasilyasRNA,DNAismorereactivetotheformalinandrequiresalonger(2-day)protease

digestiontimetoreleasesubstantialamountsofDNA.TherecoveredDNAcantypicallybeusedforPCRandotherdownstreamapplications.

Product Quantity Cat. No.TRIzol®Reagent 100mL 15596026

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Product Quantity Cat. No.

RecoverAll™TotalNucleicAcidIsolationKitforFFPE 40purifications AM1975

Figure 14.2. Overview of the RecoverAll™ Total Nucleic Acid Isolation Kit procedure.

Figure 14.3. Yield of RNA from archived human FFPE tissue samples: RecoverAll™ kit vs. two competitor systems. A10–20µmsectionfromeachoftheabovearchived human tissue blocks was isolated using each of three kits:Competitor A kit, Competitor B kit, and the Ambion® RecoverAll™ kit.Colon,lung,andbreastsampleswere1–2yearsold;kidneywas3–5yearsold;andbladderwas10–15yearsold.OncetheRNAwasisolated,thecon-centrationwasdeterminedviaOD260,andtheamountofRNArecoveredin micrograms was calculated. The RecoverAll™ Kit yielded the highestrecoveryofthethreesystemsforalltissuetypes.

Figure 14.4. Ct values from real-time RT-PCR of frozen and FFPE mouse tissues. Fourdifferenttissues(brain,kidney,heart,andliver)weredissectedfromfourdif-ferentmice.Eachtissuewassplitinhalf—onehalfwasflash-frozeninliquidnitrogenandthenstoredat–80°C,whiletheotherhalfwasfixedandembed-dedusingstandardhospitalprotocol.RNAwasisolatedfromone20µmsec-tionfromeachFFPEsampleusingtheRecoverAll™Kit.RNAfromthefrozencontrolswasisolatedusingthemirVana™miRNAIsolationKit.TheRNA(400ng)wasthensubjectedtotwo-stepreal-timeRT-PCR.ThecDNAwasgener-atedusingtheRetroScript®Kit.Ambion®SuperTaq™polymeraseandothernecessaryreagents,plusanaliquotoftheRTreaction,wereusedforthePCR.Allgenetargetshadanampliconrangingfrom80to100nt.TheCtvaluesforallreactionsofthesametissuewereaveragedtogether,thentheCtvaluesforthefrozencontrolsweresubtractedfromtheCtvaluesfortheFFPEsamplestogeneratethegraphabove.

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Protease Digestion

Nucleic Acid Isolation

Nuclease Digestion and Final Purification

20 min

3 hr or48 hr

10 min

45 min

1. Assemble FFPE sections equivalent to <–80 µm or <–35 mg unsectioned core

2. Add 1 mL 100% xylene, mix, and incubate for 3 min at 50°C

3. Centrifuge for 2 min at maximum speed, and discard the xylene

4. Wash the pellet twice with 1 mL 100% ethanol and air dry

1. Add Digestion Buffer and Protease

2. Incubate at 50°C for 3 hr for RNA isolation and 48 hr for DNA isolation

1. Add 480 µL Isolation Additive and vortex

2. Add 1.1 mL 100% ethanol and mix

3. Pass the mixture through a Filter Cartridge

4. Wash with 700 µL of Wash 1

5. Wash with 500 µL of Wash 2/3

1. Add DNase or RNase mix to each Filter Cartridge and incubate for 30 min

2. Wash with 700 µL of Wash 1

3. Wash twice with 500 µL of Wash 2/3

4. Elute nucleic acid with 2 x 30 µL Elution Solution or nuclease-free water

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5´

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Ligation of detection reagent to

3´ poly(A) tail

Hybridization tomiRNA microarray

Final wash and scanning

3´ total RNA

5´ 3´-tailed miRNA

Single reagent

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miRNA probe

Tagged miRNA

5´ 3´-labeled miRNA

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Figure 14.5. NCode™ Rapid miRNA Labeling System titration. TheNCode™RapidmiRNALabelingSystemis recommendedwhenstartingwith250ngto5μgtotalRNA.Whenstartingwithlessthan50ngoftotalRNA,werecommendusingtheNCode™miRNAAmplificationSystemtoincreasethequantityofmiRNAthatcanbedetectedandprovidesufficientmaterialforreplicateexperiments.

Figure 14.6. Sensitivity of the NCode™ Rapid miRNA Labeling System com-pared to competitor “E”. Starting with 100 ng pooled total human RNA,sampleswerelabeledwiththeNCode™Rapidorlabelingsystemfrom“E”.Replicate samples were arrayed on either NCode™ microarrays or com-petitormicroarrays inbothredandgreenchannels. ImagesscannedviaanAxonscanneranddataacquiredinaGenePixPro®scanner.Datawerebackground-correctedandcomparedacrosscommonhumanfeaturesforsignal-to-noise ratios (SNR) and positive-call designation. Positive call isdetermined as SNR >3 in all replicate features across all replicate arrays.MeanSNRwascalculated frompositivecalls. TheNCode™RapidmiRNALabelingSystemisbrighteranddetectsmorefeaturesatlowinput(100ngoftotalRNA).

Simplified miRNA fluorescent labeling

The NCode™ Rapid miRNA Labeling System is a fast, reliable method to label endogenous miRNAs with fluorescent tags. Using this system,

miRNAsfromtotalRNAsamplesarepolyadenylatedandlabeledwithfluorescentAlexaFluor®dyes,andhybridizedtomicroarraysprintedwith

species-specificantisensemiRNAprobes.ThissystemhasbeenoptimizedtoenablesensitiveandaccurateprofilingofmiRNAexpressionpatterns

fromminimalRNAinput.Startingwith250ngto5μgoftotalRNA,miRNAsaretaggeddirectlyinaquickandeasyprotocolwiththeNCode™Rapid

miRNALabelingSystem(Figure14.5).Lowerstartingamounts(100ng)maybeusedwithsimilarsensitivities,dependingonthetypeofscanner

andhybridizationmethodused.DynamichybridizationisrequiredtoachievemaximumsensitivityatlowerinputRNAlevels.Maximumsensitivi-

tiesaretypicallyseeninthe500ngto1μgrange,dependingonsampletype.Theprotocoltakesapproximately1hourtocompleteandconsists

ofjusttwostepspriortohybridization:poly(A)tailing,andligationofafluorescentmolecule.After8–16hoursofhybridization,themicroarraysare

readytoscanandanalyze.Thefluorescentdendrimer—abranchedstructureofsingle-anddouble-strandedDNAconjugatedwithAlexaFluor®

dyes—appendsapproximately15fluorophorestotheRNAstrand.Thehighsensitivityachievedwiththismethodisduetothesignalamplifica-

tioneffectofthelabeledbranchedDNAstructure[1],enablingmaximumsignal-to-backgroundratiosandstrongsignalcorrelationforincreased

sensitivity(Figure14.6,Figure14.7).

UsingtheNCode™RapidmiRNALabelingSystemenables:

→ Lesstimeatthebench—hybridizeinonestep,withnomiRNAenrichmentrequired

→ Increasedsensitivity—reliablydetectattomolarlevelsofmiRNA

→ ReliablemiRNAprofilingresults

→ Rapidturnaround—completeyourexperimentsinasingledayusingthe8hrhybridizationprotocolwithdynamichybridization

1. Thismethoduses3DNA®Reagent,manufacturedunderexclusivelicensefromGenisphere,Inc.

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>100

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NCode™ RapidCompetitor A

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Figure 14.7. Sensitivity of the NCode™ Rapid miRNA Labeling System compared to competitor “A”. Startingwith100ngpooledtotalhumanRNA,sampleswere labeledwiththeNCode™Rapid or labeling system from “A”. Replicate samples were arrayed on either NCode™microarraysorcompetitormicroarraysinthegreenchannel.ImageswerescannedviaanAxonscanneranddataacquiredinaGenePix®scanner.Datawerebackground-correctedandcomparedacrosscommonhumanfeaturesforsignal-to-noiseratios(SNR)andpos-itive-calldesignation.PositivecallisdeterminedasSNR>3inallreplicatefeaturesacrossallreplicatearrays.MeanSNRwascalculatedfrompositivecalls.Thenumberoffeatureshavingsignalsgreaterthan10,000FU(purplebars),1,000FU(darkbluebars),and100FU(redbars)areshownabove.TheNCode™RapidmiRNALabelingSystem isbrighteranddetectsmorefeaturesatlowinput.

Product Quantity Cat. No.NCode™RapidmiRNALabelingSystem 20rxns MIRLSRPD-20

NCode™RapidAlexaFluor®3miRNALabelingSystem 20rxns MIRLSA3-20

Ultrasensitive miRNA amplification

Inmanyinstances,RNAsamplesderivedfromlasercapturemicrodissection(LCM),flow-sorted(FACS)samples,orneedlebiopsiesdonotyield

sufficientstartingamountsofRNAforstandardprofilingmethodsorfortheexperimentalreplicatesneededforstatisticalreliability.Insuchcases,

it isuseful toapply linearmiRNAamplificationmethodstotheenrichedmiRNApopulation, thereby increasingtherelativetargetabundance

levels.TheNCode™miRNAAmplificationSystem,basedonmRNAamplificationmethods,enablesrobustandefficientlinearamplificationand

microarrayprofilingofsmallamountsofRNAspecies,suchasmiRNA,fromaslittleas50ngoftotalRNAortheenrichedequivalent.TheNCode™

miRNAAmplificationSystemenables:

→ Increasedsensitivity,withconsistent2,000-to5,000-foldamplificationofmiRNA

→ AmplifiedproductthatfaithfullyrepresentstheinitialmiRNAsample(Figure14.8)

→ Preciseamplificationfromreactiontoreaction

Product Quantity Cat. No.NCode™miRNAAmplificationSystem 20rxns MIRAS20

Directly labeled

Ampli�ed

let7

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Cell/tissue types

Figure 14.8. Ultrasensitive and accurate miRNA microarray profiling from small samples. Total RNA from large cell carcinomas and adjacent normal tissue was isolatedfrom multiple patients and enriched for miRNA with the PureLink™ miRNAIsolation Kit. Enriched miRNA from 300 ng of total RNA was amplified usingthe NCode™ miRNA Amplification System and labeled for array analysis withthe NCode™ miRNA Labeling System. Additionally, the miRNA from 10 μg oftotalRNAfromthesamesourcewaslabeledwiththeNCode™miRNALabelingSystemwithoutamplificationandhybridizedtoNCode™Multi-SpeciesmiRNAMicroarrays for analysis. The ratios of miRNA expression in tumor samples vs.adjacentnormaltissueswerecalculatedforallhumanmiRNA.

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Comprehensive miRNA expression profiling

WhetheryouprefertoprofilemiRNAsusingin-houseprintedarraysorpreprintedarrays,theNCode™platformallowsyoutocomprehensively

screenmostknownmiRNAsinavarietyofspecies(Figure14.9).EnjoytheadvantagesofaleadingpreprintedmiRNAprofilingmicroarray,orgain

theflexibilityofaprobesetandcontrolsforarrayfabricationinyourlab.TheNCode™microarraysoffer:

→ Arraysspottedintriplicatewithpositiveandnegativecontrolsthroughoutformonitoringhybridizationspecificity

→ Increasedsensitivityfrommaximumhybridizationintensities,andnormalizedmeltingtemperaturesforuniformhybridization

→ Easynormalizationofsignalintensitiesduringscanning,usingAlexaFluor®dyecontrolprobes

→ DetectionandidentificationofmiRNAsconservedinotherspeciesbutnotyetvalidatedforyourmodel

NCode™ Human miRNA MicroarrayTheNCode™HumanmiRNAMicroarrayV3consistsofCorning®epoxide–coatedglassslidesprintedwithoptimizedprobesequencesthattarget

nearlyallknownhumanmiRNAsintheSangermiRBaseSequenceDatabase,Release10.0,plus373novelputativehumanmiRNAs(Table14.1)dis-

coveredthroughdeepsequencingandvalidatedbybothmicroarrayhybridizationsandqPCRanalysis.Eachmicroarrayslidecomesfullyblocked

andreadytouse.Accesstothisputativenovelcontentenablesresearcherstoaccessandprofilebiologicallyrelevantsmallnon-codingRNAs

(putativemiRNAs)thatarenotavailableonotherplatforms(Figure14.10).Thiscontentisnotderivedfromin silico predictions.

NCode™ Multi-Species miRNA MicroarrayTheNCode™Multi-SpeciesmiRNAMicroarrayV2consistsofoptimizedprobesequencestargetingnearlyalloftheknownandpredictedmaturemiR-

NAsintheSangermiRBaseSequenceDatabase,Release9.0,forhuman,mouse,rat,D. melanogaster, C. elegans,andzebrafishsequences(Figure14.11,

Table14.1).Thesespecies-specific,unmodifiedoligonucleotidesare34to44basesinlength,andeachmicroarrayslidecomesfullyblockedandready

touse.InterrogationofsampleswiththeNCode™Multi-SpeciesmiRNAMicroarrayallowsresearcherstocomparecross-speciesmiRNAexpression,and

validatetheexistenceofpredictedmiRNAspecies(orthosethatareconservedbutnotyetvalidatedinaparticularorganism).

ControlsTheNCode™Multi-SpeciesmiRNAMicroarrayControlV2isasynthetic22-nucleotidemiRNApositivecontrolthatisusedtoassesstheefficiency

ofarray labelingandhybridization.Thiscontroldoesnotexhibitanydetectablecross-hybridizationor interferencewithendogenousmiRNAs

fromanysequencedorganism.TheNCode™Multi-SpeciesmiRNAMicroarrayandNCode™miRNAMicroarrayProbeSetsincludeoligonucleotide

probesthatarecomplementarytothecontrol.

Undifferentiated huESC

Liver

Ovary

Heart

Lung

Thymus

Stomach

Breast

Colon

Brain

Tissue classi�cation

Fold change

–6 0 +6

Figure 14.9. Profiling and cluster analysis of total RNA from various tissues on the NCode™ Human miRNA Microarray V3. Bothknownandnovelcontentshowtissue-specificdifferentialexpression.

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36532869 (hsa-miR-758)2121 (rno-miR-499|mmu-miR-499|bta-miR-499)2124 (mmu-miR-124a)320436403312363736223643336833593534337833433363349233721755 (mmu-miR-451|rno-miR-451|dre-miR-451|xtr-miR-451)3365297734491851 (rno-miR-422b)25841498 (mmu-miR-133b|hsa-miR-133b|gga-miR-133b|dre-miR-133b|rno-miR-133b|xtr-miR-133b)33621311 (mmu-miR-195|hsa-miR-195|rno-miR-195|ggo-miR-195|ppa-miR-195)2262 (dre-miR-1|fru-miR-1|tni-miR-1)1385 (mmu-miR-150|hsa-miR-150|rno-miR-150)2340 (dre-miR-133a|fru-miR-133|tni-miR-133)2005 (mmu-miR-130a|hsa-miR-130a|rno-miR-130a|dre-miR-130a|xtr-miR-130a)3369352432392563

Fold change

–6 0 +6

avg

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Figure 14.10. Novel putative miRNA content in adenocarcinoma. The NCode™ HumanmiRNAMicroarrayV3wasusedtoprofileadenocarcinomasamples,andprofileswerecompared to matched normal tissue. Significant expression changes were seenwithinthenovelputativemiRNAcontentonthearray, inadditiontotheexpectedknownmiRNAsfromtheSangermiRBase.

Subarray 1Replicate 1 of 3



Table 14.1. Number of probe sequences in the NCode™ miRNA microarrays.

Probe content Human miRNA array V3 probes Multi-species miRNA array V2 probes

BasedonSangermiRBaseSequenceDatabaserelease Version10.0 Version9.0

Human 710plus373putative 553

Mouse 427

Rat 261

C. elegans 115

D. melanogaster 85

Zebrafish 371

SmallnucleolarRNAs 29 10

Mismatchcontrols 76 76

NCode™positivecontrol 1(72)* 1(72)*

NCode™dyenormalizationcontrols 5 5

*TheNCode™positivecontrolisspottedthroughoutthreesubarrays(72wells).

Product Quantity Cat. No.NCode™HumanmiRNAMicroarrayV3 5arrays MIRAH305

NCode™Multi-SpeciesmiRNAMicroarrayV2 5arrays MIRA2-05

NCode™Multi-SpeciesmiRNAMicroarrayProbeSetV2 500pmol MIRMPS2-01

NCode™Multi-SpeciesmiRNAMicroarrayControlV2 10μL MIRAC2-01

NCode™HumanmiRNAMicroarrayProbeSetV3 500pmol MIRHPS301

Figure 14.11. NCode™ miRNA microarray content. Each epox-ide slide contains probes for profiling designated miRNAsequences, spotted in triplicate. NCode™ probes for theNCode™miRNAMicroarrayControlareprintedthroughoutthearraytofacilitateanalysis,asaremismatchandshuffledcontrolsformonitoringhybridizationspecificity.

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ProbesForresearchersplanningtospottheirownmiRNAmicroarrays,InvitrogenhasprobesfromtheNCode™HumanmiRNAMicroarrayV3andNCode™

Multi-Species miRNA Microarray V2 available in 384-well plates. Each well contains 500 pmol of lyophilized oligonucleotide probe, ready for

resuspensionandprinting.

Simplified data analysis using the NCode™ ProfilerUse of Latin squares loop design/dye swap methodology. Thereareanumberofmethodsusedtoanalyzeexpressionprofilingmicroarraydata,

butsomearenotidealformiRNAmicroarraysduetothereducednumberoffeaturespresentandthedynamiclevelsofmiRNAexpressionacross

samples.WerecommendusingtheLatinsquaresnormalizationandloopdesign/dyeswapmodeldescribedbyKerretal.[3],becausethismethod

providesreliablestatisticaldatawiththesmallestnumberofmicroarraychips.Thisexperimentaldesigngeneratesaloopwithoutidenticalchip

replicates,thougheachtissuewillbereplicatedtwice,oncewitheachAlexaFluor®dye.Usingthisarraylayoutenablescomparisonofeachtest

samplewithboththereferencesampleandcomparisonofeachtestsamplewithanyothertestsample.Thisnormalizationmethodattemptsto

reduceoreliminateanygeneralmiRNAeffect,arrayeffect,dyeeffect,overalltissueeffect,array–miRNAinteraction,anddye–miRNAinteractions

fromthedata(Figure14.12).

Experimental design. NCode™ProfilersoftwareenablessimpledesignandanalysisofmiRNAmicroarraydatausingloopdesignanddyeswap

normalizationmethodsthatsupportsample-to-samplecomparisonwithintheexperiment.Theyaredesignedwithstatisticalconsiderationsto

reduceoreliminateanygeneralmiRNAeffect,arrayeffect,dyeeffect,overalltissueeffect,array–miRNAinteraction,anddye–miRNAinteraction

fromthedata.

Data analysis. miRNAresearchersnowhaveatailoredtoolto identifydifferentiallyexpressedmiRNAmarkersonmicroarrays.NCode™Profiler

softwareisanadvancedexperimentaldesignandanalysissolutiondesignedfortwo-dyeexpressionprofilingmicroarrayexperiments.Oncean

experimenthasbeenconducted,therawdatacanbe importedandanalyzedusingthestatisticalmethodsdesignedintothearray.Foreach

tissueonthearray,pairwisedifferentialexpression isdeterminedandtheteststatistics, foldexpressionchanges,andp-valuesaregenerated.

Additionally,therankingofeachmiRNAwithrespecttotheothermiRNAsintermsofoverallexpressionlevelwithinthetissueisgiven.Using

NCode™Profilersoftwareenables:

→ Simplifiedexperimentaldesignsteps

→ Confidenceinterpretingresultswithprovenstatisticalanalysisusingdyeswaporloopdesignnormalizationmodels

→ Outputincludesteststatistic,foldexpressionchangesforeachpair-wisecomparison,andp-valuesandrankingsofmiRNAmarkersforall

samples

→ Easyexportofnormalizeddatatovisualizationsoftwareforclustering(tree)andheatmapanalysis

Visitwww.invitrogen.com/ncodetodownloadafreecopy.NCode™ProfilersoftwarerunsonMicrosoftWindows®XPandWindowsVista®operatingsystems.

Figure 14.12. The NCode™ Profiler software for miRNA microarray data analysis has a simple user interface and provides both data analysis using Latin squares nor-malization and experimental design guidance.

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Robust data validation using the NCode™ miRNA qRT-PCR KitsQuantitativeRT-PCR(qRT-PCR)isthestandardforvalidatingmicroarraydataandisaninvaluabletoolforhighlysensitiveandaccurateprofilingof

miRNApopulationsubsets.MostcommerciallyavailablemiRNAqRT-PCRsystemsuseproprietary,predesigned,miRNA-specificprimersforreal-

timePCRand/orreversetranscription.Unfortunately,suchprimersrequirethatthemiRNAsequenceispubliclyavailable,andthatacommercial

qRT-PCRassayhasbeendevelopedforthatspecificsequence.ThislimitstheavailabilityofqRT-PCRassaysformanymodelorganisms,recently

discoveredmiRNAsornon-codingRNAs,orproprietarymiRNAsequences.

The NCode™ VILO™ miRNA cDNA Synthesis and NCode™ EXPRESS SYBR® GreenER™ miRNA qRT-PCR Kits overcome these limitations by

combiningacarefullyoptimizedcombinedpolyadenylationandreversetranscriptionstepusingtheSuperScript®VILO™enzymeandauniversal

primer.ThemiRNA-specificamplificationoccursduringthePCRreaction,inwhichthesequenceofthemiRNAofinterestisusedasthetarget-

specificPCRprimer.

UsetheNCode™EXPRESSSYBR®GreenER™miRNAqRT-PCRKitsto:

→ AmplifyusingminimaltotalRNAinput(noadditionalenrichmentneeded,conserveprecioussamples)(Figure14.13)

→ DetectmiRNA,ncRNA,ormRNAsequencesfromthesameuniversalcDNA

→ ArchivecDNAfor latervalidation—theuseof thecDNAformultipleexperimentswillenablemorereliablecomparisonandallowfuture

interrogationofnewsequences

→ ObtainexcellentsensitivityoverabroaddynamicrangeofmiRNAabundance

→ ProfilecloselyrelatedmiRNAswithsingle-nucleotidediscrimination

→ Usemultiplereal-timePCRinstrumentplatforms,includingstandardandfast-cyclingmodes

Figure 14.13. miRNA detection from total RNA or enriched miRNA starting materials. cDNAwasgeneratedfrombrainreferenceRNAoranequivalentvolumeofenrichedmiRNA.AverageCtvaluesfor10ng/µLtotalRNAorequivalentperqPCRreactionareshown.

MicroRNA Total RNA Enriched RNA

26a 16.53 16.53

195 17.19 17.28

1 24.97 25.36

Match the NCode™ qPCR tool to your research needs

NCode™ EXPRESS SYBR® GreenER™ miRNA qRT-PCR Kits—incorporateprovenInvitrogenenzymesandthenewestSYBR®GreenER™dyespecifi-

callytailoredforsuperiorresultsonhigh-throughput,fast-cyclinginstruments.Thesekitsprovide:

→ Fast-activating,antibody-mediatedPlatinum®TaqDNAPolymeraseinassociationwiththebrighterandlessPCR-inhibitorySYBR®GreenER™

dye—complete,rapidactivationthatenablesthebestsensitivityandreproducibility

→ FlexibleformatwithpremixedorseparateROX—designedtoworkwithdefaultinstrumentprotocolsonawiderangeofhigh-throughput

fastinstruments(e.g.,AB7500FAST,AB7900HTFast,ABStepOne™System,RocheLightCyler®480,CorbettRotor-Gene™

→ SignificantlyreducedcarryovercontaminationwithUDGcarryoverprotection—uracil-DNA-glycosylase(UDG)/dUTPincorporatedinallkits

3020100 40

miR-26a

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NCode™ VILO™ miRNA cDNA Synthesis Kit—providesreagentsonlyforthefirst-strandcDNAreactionallowingyoutouseyourownoptimized

qPCRsupermix.TriedandtestedSuperScript®IIIReverseTranscriptaseisformulatedinanenhancedbuffersystemtoprovideyouwiththemost

reliablefirst-strandsynthesisandhighercDNAyields.

Product Quantity Cat. No.NCode™VILO™miRNAcDNASynthesisKit 50rxns A11193050

NCode™EXPRESSSYBR®GreenER™miRNAqRT-PCRKitWithPremixedROX 200rxns A11193052

NCode™EXPRESSSYBR®GreenER™miRNAqRT-PCRKitUniversal 200rxns A11193051

Figure 14.14. The NCode™ Long Non-coding RNA Database.

NCode™ Non-coding RNA Arrays

Accelerate discovery with the first commercially available microarrays for profiling long non-coding RNA (ncRNA)

NCode™HumanandMouseNon-codingRNAMicroarraysarefirst-generationhigh-densityarraysdesignedtoprofilelongerncRNA(>200bases)

andtoanalyzemRNAsimultaneouslyonthesamearray.InadditiontothencRNAcontent,probestargetingmRNAallowdiscoveryofcoordinated

expressionwithassociatedprotein-codinggenes.

NCode™ Long Non-coding RNA DatabaseTheNCode™LongncRNADatabasecontainsinformationrelatingtothelongncRNAandmRNAsequencesprofiledbytheNCode™Humanand

MouseNon-codingRNAArrays(Figure14.14).The17,000+long(>200bases)ncRNAsfromhumanand10,000+ncRNAsfrommousearepredomi-

nantlyderivedfrompublishedsources.Searchprobe-specificinformationforthencRNAMicroarraybyselectingspeciesandtheSearch,Browse,

orBLASTfunctions.

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ProfilencRNAwithyourexistingexpressionarrayworkflow

→ UsingtheNCode™microarrays,youcan:

→ Profilemorethan17,000long(>200bases)ncRNAsfromhumanandmorethan10,000ncRNAsfrommouse,derivedfrompublishedsources*

(Table14.2)

→ Correlate ncRNA expression with mRNA expression for known pathways, with simultaneous profiling of coding and non-coding RNAs

(Figure14.15)

→ PerformncRNAresearchusingstandardexpressionarraylabelingtechnologiesandinstrumentation–includingtheAgilent®Technologies

system

TheNCode™HumanandMouseNon-codingRNAMicroarraysareavailableinaduplexarrayformatintwosizes(1slideand5slides).

Figure 14.15. NCode™ Human Non-coding RNA Array permits simultaneous detection of mRNA and ncRNA expression in biological samples. ThehistogramdescribestheobservedfoldchangeofbothmRNAandncRNAtranscriptsinbreastcancertumorsamplesvs.adjacentnormaltissuesinasinglepatient.Foldchangefromexpressionlevelsfromnormaltissuewerecalculatedforknownbreastcancerassociatedtranscripts(bothcodingandnon-coding)derivedfromthetumorgene.orgdatabase.Non-codingsequenceswereidentifiedbyanalgorithmthatscoresvariouscharacteristicsofprotein-codinggenesincludingopenreadingframelength,synonymous/nonsynonymousbasesubstitutionrates,andsimilaritytoknownproteins[1].AlongwithputativelongncRNAs,thearrayalsotargetscharacterizedncRNAs,suchasXist,Air,PINC,Neat1,andNeat2.AlthoughthearraytargetssomemiRNAprecursors,itisnotdesignedtodetectmiRNAs.RefertotheNCode™miRNAproductsfortoolsoptimizedformiRNAlabeling,arrays,andqRT-PCR.

Table 14.2. Each slide contains non-coding RNA and mRNA features and controls for monitoring experimental success.

Format Human ncRNA Array Mouse ncRNA Array

Totalfeatures22,074codingfeatues17,112non-codingfeatures*

25,179codingfeatues10,802non-codingfeatures*

Controlfeatures 1,325oneacharray 1,325oneacharray

*ThencRNAprobespredominantlytargetfull-lengthcDNAscatalogedfrompublishedstudies.Specifically,thencRNAcontenthasbeendevelopedfromthefollowingsources:manualcurationfromliterature,FunctionalAnnotationofMouse(FANTOM3)project,HumanFull-lengthcDNAAnnotationInvitational(H-Invitational)project,antisensencRNAsfromcDNAandESTdatabaseformouseandhumanusingacomputationpipeline[4],humansnoRNAsandscaRNAsderivedfromsnoRNA-LBME-db,RNAz[5],Non-codingRNASearch[6],andEvoFold[6].

Product Quantity Cat. No.NCode™HumanNon-codingRNAMicroarray 1slide NCRAH-02

NCode™HumanNon-codingRNAMicroarray 5slides NCRAH-10

NCode™MouseNon-codingRNAMicroarray 1slide NCRAM-02

NCode™MouseNon-codingRNAMicroarray 5slides NCRAM-10

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Genome-wide analysis of long non-coding RNA (ncRNA) expression in solid tumors using the NCode™ Non-coding RNA Microarray platformNumerousstudieshavedemonstratedthatinhigherorganisms,mostofthenonrepetitivegenomeistranscribedinadevelopmentallyregulatedfash-

ion.Interestingly,onlyasmallpercentage(<20%)ofthesetranscriptsareassociatedwithgenesthatencodeproteins[1].Non-codingtranscriptscome

inawiderangeofsizes(18ntto100kb),and,instarkcontrasttowhatisknownofprotein-codingtranscripts,veryfewnon-codingtranscriptshave

experimentallyderivedfunctions.Recentevidencesuggeststhatthousandsoflongnon-codingRNAsareexpressedwhosefunctionshaveyettobe

studiedbutwhichappeartoplayimportantrolesingeneregulation.Microarraystudieshavedemonstratedthatamajorityofthemammaliangenome

istranscribed[8],oftenonbothstrands,toproduceoverlappingandinterlacingtranscripts,manyofwhicharecell-andtissue-specific.Whilemostareas

yetunstudied,increasingevidencesuggeststhesenon-codingtranscriptsaffectmanyofthedevelopmentalandregulatoryprocesses[9]thatarecritical

incancerresearchinvestigations.Morerecentdataonlargeinterveningnon-codingRNAs,orlincRNAs[10],furtherdefineagroupoffunctionalncRNAs

thatareconservedandimplicatedindiversebiologicalprocesses.HerewedescribetheutilityoftheNCode™Non-codingRNAMicroarrayplatformfor

simultaneouslyprofilingcodingandnon-codingtranscriptsfromthreetypesofsolidtumorscomparedtoadjacentnormaltissue.

New microarrays to examine ncRNAsWith the maturation of next-generation sequencing technologies, more ncRNA transcripts, especially the longer classes, are becoming the

subject of gene expression research. In 2008, two novel microarrays were launched to assist researchers in the study of ncRNA biology: the

NCode™HumanandMouseNon-codingRNAMicroarrays.Thesetwohigh-densityarrayscontaincontentdevelopedbyJohnMattick(University

of Queensland, Australia), using algorithms that identified non-coding transcripts present in public cloning and sequencing databases like

FANTOM3.Thehumanarraycontainsprobestargeting17,000uniquenon-codingtranscriptsfromvariousdatabasesand22,000uniqueprotein-

codingtranscriptsfromtheRefSeqcollection,printedinduplicatealongwiththousandsofstandardpositiveandnegativecontrols.Themouse

arraycontainsfeaturesfor10,800uniquenon-codingtranscriptsand23,000uniqueprotein-codingtranscriptsfromtheRefSeqcollection,also

printedinduplicatewiththestandardpositiveandnegativecontrols(Figure14.16).

NCode™ Human and Mouse ncRNA MicroarraysDatapresentedherewereobtainedusingtheInvitrogenNCode™HumanNon-codingRNAMicroarrayplatform(Cat.No.NCRAH10).Thishigh-density

microarrayisthefirstcommerciallyavailabletoolcapableofsimultaneouslyprofilingbothlongnon-codingRNA(ncRNA)expressionandmessenger

RNA(mRNA)expressionfromasinglesample(Table14.3).TheNCode™HumanNon-codingRNAMicroarrayismanufacturedwithAgilentSurePrint®

technology,andisprintedina2x105Kformat,whichallowsfortwoindividualsamplehybridizationsperslide.Non-codingsequenceswereidentified

byaproprietaryalgorithmthatscoresvariouscharacteristicsofprotein-codinggenes,includingopenreadingframelength,synonymous/nonsynony-

mousbasesubstitutionrates,andsimilaritytoknownproteins[1].CharacterizedncRNAsmanuallycuratedfromliteratureaswellasfrompublicdatabase

repositoriesarealsoincluded.InadditiontothencRNAcontent,probestargetingmRNAcontentfromtheRefSeqdatabaseandothercollectionsarealso

included,allowingdiscoveryofcoordinatedexpressionwithassociatedprotein-codinggenes.

Data analysisThemicroarrayswerescannedwithanAgilentDNAMicroarrayScanner,andFeatureExtractionSoftwarewasusedtogeneraterawdatafiles.Rawdata

wereuploadedintoGeneSpringGX7.3.1Software(Agilent)fornormalizationandanalysis.Normalizationwasperformedusingthedefaultparameters

forAgilentone-colorarraydata.Expressionlevelsofdifferentiallyexpressedtranscripts,bothcodingandnon-coding,werevalidatedusingtheEXPRESS

SYBR®GreenER™qPCRSuperMix(InvitrogenCat.No.11784-200).Thresholdcycle(Ct)valueswerenormalizedtotheGAPDHhousekeepinggene.

NCode™ Human Non-coding RNA ArrayVersion 1.0 Content

NCode™ Mouse Non-coding RNA ArrayVersion 1.0 Content

mRNA-associated22,074

Array controls1,325

ncRNA-associated17,112

mRNA-associated25,179

Array controls1,325

ncRNA-associated10,802

Figure 14.16. Charts representing content of the Human and Mouse Non-coding RNA Microarrays, highlighting the ncRNA and mRNA percentages.

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Evaluation of labeling strategies for NCode™ ncRNA array analysis AsavalidationoftherecommendedRNAlabelingapproaches,wecomparedRNAexpressionprofilesfromalungtumorsamplelabeledusing

cDNAsynthesisprimedwitheitheranoligo(dT)primer,similartotheprimerusedintheSuperScript®IndirectRNAlabelingmethod,orrandom

primers,oracombinationofboth.Thisallowedustoexaminetheefficacyofusingapoly(A)tail–targetedprimingmethodtoanalyzencRNA

transcripts,manyofwhicharethoughtnottobepoly(A)-tailed.AscanbeseeninFigure14.17,morencRNAfeaturesweredetectedwitholigo(dT)

primingthanwithrandomprimingorwithrandomprimingincombinationwitholigo(dT)priming.Additionally,ourcomparisonrevealedvery

highPearsoncorrelations(r2=0.87and0.90,respectively)betweenpoly(A)-primedsamplesandrandomlyprimed,orrandomlyprimedinaddi-

tiontooligo(dT)-primedsamples.Thesedataindicatethatalthoughmanyofthesetranscriptsmaynothavepoly(A)tails,themajorityarestill

detectablebypoly(A)-specificprimingduetointernalpoly(A)sequencestretches.Thisisnotsurprisinggiventhatthenon-codingcontentwas

minedfrompublicexpressionlibrarydatabasesthatweregeneratedusingoligo(dT)-primedcDNAsynthesismethods.Wealsoexaminedthe

normalizedsignalintensitiesofasubsetoftranscripts(Figure14.18)andobservedthatboththeoligo(dT)-primedcDNAprobesandoligo(dT)-

primedplusrandomlyprimedcDNAprobesprovidedsimilarsignalintensities.

Table 14.3. Non-coding and coding-associated transcripts targeted in the NCode™ microarrays.

Number of unique sequences (60-mer, printed in duplicate)

Number of putative long non-coding RNAs (>200 nt) targeted

Number of coding-associated transcripts targeted

NCode™HumanNon-codingRNAMicroarray*

39,186 17,112 22,074

NCode™MouseNon-codingRNAMicroarray*

35,981 10,802 25,179

*1,325Agilentpositivecontrolfeaturesarealsoincludedineacharray,andareusedforgeneratingautomatedQCreports.

Non

-cod

ing

RN

A fe

atur

es d

etec

ted

Oligo(dT)/randomhexamer

Randomhexamer

Oligo(dT)0

750

1,500

3,000

4,500

6,000

2,250

3,750

5,250

Priming method

3,934

2,534

3,148

Oligo(dT) Random hexamer

Oligo(dT)/ random hexamer

Oligo(dT) 1.00

Random hexamer 0.87 1.00

Oligo(dT)/ random hexamer

0.90 0.94 1.00

Figure 14.17. Comparison of RNA labeling strategies. ThehistogramshowstheoverallnumberoftranscriptsdetectedusingcDNA-labeledprobesgeneratedwitholigo(dT)-primedvs.randomlyprimedcDNAtemplates.Additionally,thetableshowsthePearsoncorrelationbetweendatasetsfromeachprimingmethod.

6,000

5,000

4,000

3,000

2,000

1,000

0

ncRNA

AK123891 AY927614 BX538013 BC071805AF086201 uc001vxa BC127836 uc003ixr

Nor

mal

ized

inte

nsity

Oligo(dT)

Random hexamer

Oligo(dT) + random hexamer

Figure 14.18. Relative signal intensities of different cRNA labeling strategies. Thehistogramsaboveshowtherelativesignal intensitiesofanumberofncRNAtranscripts,detectedassignificantlyabovebackground,witheachprimingmethod.

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Figure14.19showsthegeneralizedworkflowfromRNAisolationtoresultsvalidation.cDNAprobesweregeneratedusingtheSuperScript®

IndirectRNAAmplificationSystem(InvitrogenCat.No.L1016-02)accordingtothemanufacturer’sprotocol,using1μgoftotalRNAineachlabel-

ingreaction.Threematchedpairsofhumanprimarytumorsandadjacentnormaltissuewereprofiled.TotalRNAisolatedfrombreast,colon,and

lungtissue (obtained fromBioChain,Hayward,CA)wasextractedwithTRIzol® reagent (InvitrogenCat.No.15596026).A1,000-folddilutionof

AgilentOne-ColorRNASpike-InMix(containingexogenousmRNAtranscriptsthatarespecifictotheAgilentcontrolfeaturesfoundontheAgilent

array)wasaddedto1μgoftotalRNAsampleineachtube.TheRNAsampleswereamplifiedandlabeledusingtheSuperScript®IndirectRNA

AmplificationSystemfollowingthesuggestedprotocol.TheresultingaminoallylcRNAwaspurifiedandindirectlylabeledwithAlexaFluor®555

reactivedye(InvitrogenCat.No.A32756)usingthesuppliedprotocol.ThelabeledcRNAwasquantitatedusingaNanoDropinstrument.Additional

RNAfromeachsamplewaslabeledusingtheSuperScript®IndirectcDNAlabelingmethod,primedwitheitheroligo(dT)orrandomprimers.The

GeneExpressionHybridizationKit(Agilent)wasusedtoprepare1.5µgoflabeledcRNAfollowingthesuggestedprotocol.Hybridizationbuffer

(2X)wasaddedtothefragmentedsamples,andcarefullyloadedontothearrayusingHybridizationGaskets(AgilentPartNumberG2534-60002)

andSureHybchambers(bothfromAgilent).Afteranovernighthybridizationat65°C,thearrayswereprocessedusingAgilentwashbuffers.

Profiling mRNA and ncRNA expression in solid tumors with the SuperScript® Indirect RNA Amplification SystemAlloftheprimarysolidtumorcomparisonsyieldedspecificrelativeexpressionchanges(comparedtoadjacentnormaltissue)thatcanbeasso-

ciatedwithtumorigenesis,aswellasdistinctlytissue-specificexpressionprofilesnotassociatedwithdiseasestatebutrathertissuetype.Solid

tumor–specifictranscripts,bothcodingandnon-coding,weredetectedfrommanygenomiclociinboththesenseandanti-senseorientations

(Figure14.20).Theannotationfilessuppliedwiththemicroarrayallowedcomplexgenomiclocationanalysisofspecificchromosomallocations

thatcouldbeassociatedwithrelativechangesinexpression.

RNA isolation HybridizationLabelingScanning and data analysis

qRT-PCR validation

<5 µgtotal RNA

TRIzol® reagent

ncRNA arrayAgilent Spike-In ControlAgilent Hybridization/

Wash Buffer Kit

Agilent scanner, software of choice

EXPRESS SYBR® GreenER™ qPCR

SuperMixSuperScript® Indirect RNA

Amplification System

Figure 14.19. Recommended workflow from RNA isolation to qRT-PCR validation of microarray data.

Figure 14.20. Profiling mRNA and ncRNA expression in three tumor types. The heat maps and genomic maps depict changes and locationofbothcodingandnon-codingtranscriptiononchromosome17associatedwitheachtissueandtumortype.Thespecificgenometractsdisplayedcomparepubliclyavailabletranscriptomeannota-tionstoourobservedexpressionprofiles.

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Togainamoredetailedpictureofglobalchangesingeneexpressionassociatedwithprimarytumors,effortswerefocusedongeneexpres-

sionprofilesinprimarybreasttumortissuecomparedtoadjacentnormaltissue.ThescatterplotinFigure14.21showsglobalexpressionofncRNAs

andmRNAsinbreasttumorcomparedwithadjacentnormalbreastepithelialcells.Ascanbeseenfromthisgraph,themajorityofbothcodingand

non-codingtranscriptsshowequivalentlevelsofexpressionbetweenthetwosamples;however,thereisclearevidenceofdifferentialexpression

forbothtypesoftranscripts,bothup-anddown-regulated,inthetumorsample.Overall,1,155non-codingtranscriptsand2,281mRNAtranscripts

showedstatisticallysignificantdifferencesinexpressionlevelsinthebreasttumorsample(Figure14.22).

Furthermore,differentialexpressionofaknownbreastcancer–specificmRNAmarker,HER2,wasseen.Specifically,a3-foldup-regulationof

HER2 transcript in the tumor sampleswasobserved,which isconsistentwith thepreviously reportedobservation thatHER2 isup-regulated in

~15–20%ofbreastcancertypes[8].Interestingly,wealsodetectedsignificantchangesinapreviouslyunidentifiedncRNAtranscriptinthevicinityof

theHER2gene(arrayprobeID:IVGNh21558)(Figure14.23).

Adjacent normal breast tissue(normalized intensity)

Bre

ast

prim

ary

tum

or t

issu

e(n

orm

aliz

ed in

tens

ity)

0.01 0.10 1 10 1,0001000.01

0.10

1

10

100

ncRNA (2SD > BG)mRNA (2SD > BG)ncRNA (±2-fold vs. normal)

Figure 14.21. Differentially expressed transcripts in breast tumors. Thescatterplotabovedepicts (1) ncRNA expression within at least two standard deviations above back-ground in primary breast tumor and/or adjacent normal breast tissue (black), (2)mRNA expression within at least two standard deviations above background inprimarybreast tumorand/oradjacentnormalbreast tissue (blue),and (3)changesin ncRNA expression greater than 2-fold up-regulated (red) and down-regulated(green). This genome browser view represents the relative change and genomiclocationfortheHer2/neugeneandsurroundingareas.

Figure 14.22. Differentially expressed transcripts in breast tumors. Thehistogramdepictsthecodingandnon-codingtranscriptsexhibiting>2-foldup-ordown-regulationinbreasttumorvs.normalbreasttissue.

Num

ber

of p

ositi

ve fe

atur

es

mRNAncRNA

500

0

1,000

1,500

2,000Up-regulated features

Down-regulated features

Feature type

388

767 736

1,545

Figure 14.23. Differentially expressed genes in the vicinity of HER2. ThisfiguredepictstherelativeexpressionandgenomiclocationfortheHER2geneandsurroundingareas.Thelevelsofup-regulationofHER2expressionandtheadjacentncRNAtranscriptareshown.

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AsshowninFigures14.24and14.25,differentiallyexpressedtranscripts,bothcodingandnon-coding,werevalidatedusingtheEXPRESS

SYBR®GreenER™qPCRSuperMix (InvitrogenCat.No.11784-200).Most importantly,differentialexpressionof theHER2geneand theadjacent

ncRNAtranscriptwasconfirmedtobeup-regulatedinbreasttumors.

ConclusionsThe importance of non-coding RNA (ncRNA) in cellular functions, development, and disease has only been recently appreciated, but is now

widely accepted. One of the critical issues hindering the elucidation of ncRNA function is the lack of research tools to study them. Here we

describetheuseofahigh-densitymicroarraytoprofiletheexpressionpatternsof17,000ncRNAsinhumans,whichshouldhelpexpeditethis

functionalinvestigation.Inaddition,thisarraycontainsfeaturesfor22,000mRNAs,allowingresearcherstosimultaneouslyprofilebothcodingand

non-codingRNAexpressionintheirmodelsystems.

Wedescribetheprofilingofthreesolidtumorsfromthreeseparatepatientsandshowthepowerofthisarrayforidentifyingdifferentially

expressedtranscripts,bothmRNAsandncRNAsassociatedwiththesecancers.Inoneprimarybreasttumor,weconfirmtheoverexpressionofthe

HER2gene,whichhasbeendescribedpreviously.Interestingly,wealsoidentifyanovelncRNAtranscriptdownstreamoftheHER2genethatis

alsoup-regulatedinthisbreasttumor.AlthoughthefunctionofthispreviouslyunknownncRNAtranscriptanditsrelatednesstoHER2expression

andfunctionarenotknown,researchersusingstandardmRNAexpressionarrayswouldnothavebeenabletoevendiscoverthistranscript.This

demonstratesthepowerofthisnovelarrayproduct.Subsequentstepsinthisworkflowmightincludetheexaminationoffunctionbyup-and

down-regulationofspecificsequencestostudyendogenousorreportergeneregulationandphenotypicresponse.

mRNAuc001qfd

mRNACR592336

mRNAAK125544

mRNAuc002fwd

mRNABC068477

ncRNAAK091224

1,000

100

10

1

4.6 5.0

164.5

54.0

2.33.4

39.5

3.4

12.1

4.2 4.5 3.8

qPCR

Array

ERBB2/HER2

Fold

cha

nge

rela

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to n

orm

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Chapter references1. MercerTR,DingerME,SunkinSMetal.(2008)Proc Natl Acad Sci U S A 105:716–721.

2. ChomczynskiP,SacchiN(1987)Anal Biochem162:156–159.

3. KerrMKet.al(2000)J Comput Biol7:819–837.

4. RossJS,FletcherJA,LinetteGPetal.(2003)Oncologist8:307–325.

5. MattickJS,MakuninIV(2006)Hum Mol Genet15(SpecNo1):R17–29.

6. GuttmanM,AmitI,GarberMetal.(2009)Nature458:223–227.

Additional references for genome-wide analysis of long non-coding RNA (ncRNA) expression in solid turmors using the NCode™ Non-coding RNA Microarray platform

AmaralPP,DingerME,MercerTRetal.(2008)Science319:1787–1789.

CarninciP,KasukawaT,KatayamaSetal.(2005)Science309:1559–1563.

DingerME,AmaralPP,MercerTRetal.(2008)Genome Res18:1433–1445.

DingerME,MercerTR,MattickJS(2008)J Mol Endocrinol40:151–159.

MehlerMF,MattickJS(2007)Physiol Rev87:799–823.

RinnJL,KerteszM,WangJKetal.(2007)Cell 129:1311–1323.

YuW,GiusD,OnyangoPetal.(2008)Nature451:202–206.

Figure 14.25. NCode™ qRT-PCR validation of HER2/neu and adjacent ncRNA. ThefigureaboveshowstherelativeexpressionofHER2/neumRNAandadjacentncRNAasmeasuredbyqRT-PCRandmicroarray.

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Section III—miRNA Profiling

Chapter 15—Introduction to DNA methylation . . . . . . . . . . . . . . . . . . . 134


Section III miRNA Profiling

CHAPTER 15

Introduction to DNA methylation

Overview of DNA methylation

DNAmethylation isaDNAmodification thatoccursnormally inbotheukaryoticandprokaryoticorganisms. Inmanyplantsandanimals, it is

characterized by the biochemical addition of a methyl group (–CH3) to the cytosine C5 in cytosine-phosphate-guanine (CpG) dinucleotides

viaamethyltransferaseenzyme[1].Inplants,thecytosinecanbemethylatedintheCpG,CpNpG,andCpNpNcontexts,whereNrepresentsany

baseexceptguanine(Figure15.1).AlthoughCpGdinucleotidesoccurratherinfrequentlyinmammaliangenomes(approximatelyone-fourththe

expectedfrequency),DNAsegmentsabundantwithCpGdinucleotidesdoexist.CalledCpGislands,thesesegmentsaretypically100–500base

pairslongandoftencorrespondtotranscriptionstartsitesandadjacentexons.CpGdinucleotidesoccurringwithinpromotersorfirstexonsare

muchlesslikelytobemethylatedcomparedtothoseappearingelsewhere.DNAmethylationstudieshavegainedprominence,inpart,because

aberranthypermethylationofCpGislandscanoccurandhasbeenlinkedtochangesintranscriptionandgeneexpression.Theseeventshave

beenshowntoplayacentralroleincancer,geneimprinting,embryonicdevelopment,X-chromosomegenesilencing,cellcycleregulation,and

manyotherbiologicalfunctions[2,3,4].Formoreinformation,visittheepigeneticslearningcenterontheInvitrogenwebsiteatwww.invitrogen

.com/epigenetics.

DNA methylation analysis

MethodsforDNAmethylationanalysiscanbegenerallycategorizedaseitherglobalorlocus-specific.Globalmethylationanalysismeasuresthe

overalllevelofmethylatedcytosinesinasample.Locus-specificmethylationanalysisencompassesalargenumberoftechniquesthatdetermine

specificmethylationpatternsatasinglelocation(withinapromoterregion,forexample).Genome-widemethylationanalysistechniquesinclude

somemicroarraymethods,bisulfitesequencing,antibody-baseddetectionsuchasMeDIP,andDNAmethyltransferaseassays.Themostcommon

locus-specificmethylationtechniqueisbisulfiteconversionfollowedbyavarietyofPCRmethodsorbisulfitesequencingofPCRfragments.Other

techniquesincludeenzymaticdigestionfollowedbySouthernblotanalysisorPCR.MassspectrometryhasalsobeenusedforDNAmethylation

analysis todeterminespecificmethylationpatterns.As thenext-generationsequencingtechnologymatures,newmethods for locus-specific

methylationdetectionareeagerlyanticipated.

Methylated DNA enrichment

Highly sensitive enrichment of methylated DNAMethylMiner™ Methylated DNA Enrichment Kit enables superior enrichment and differential fractionation of double-stranded DNA based on

CpGmethylationdensity,with increasedsensitivityoverantibody-basedmethods (Figure15.2).Fractionationpermits importantcomparisons

betweensamplesandenablesresearcherstofocusanalysisononlythemethylationdensitiesofinterest.

AdvantagesofMethylMiner™MethylatedDNAEnrichmentKitinclude:

→ Partitioningwithhigh-affinitybinding—atleast4-foldgreatersensitivitythanantibody-basedmethods(Figure15.3)

→ FractionationbasedonCpGmethylationdensity—dsDNAcaptureisachievedwithMBD2proteinandfacilitatesligationofdouble-stranded

adaptorsfornext-generationsequencing

→ RapidandeasyelutionwithsalteliminatestheneedforproteinaseKtreatmentandphenol/chloroformextraction

→ Preciseanswers—fractionatedDNApermitsdistinctionstobemaderegardingmethylationstatusanddensity

→ Fastprotocol—completedinlessthan4hours

→ Easy—simplehandlingwithDynabeads®magneticbeads—thegoldstandardinmagneticbeads

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Product Quantity Cat. No.MethylMiner™MethylatedDNAEnrichmentKit 1kit ME10025

Figure 15.3. MethylMiner™ Methylated DNA Enrichment Kit cap-tures more heavily methylated DNA compared to an antibody-based method. Relative capture and recovery of qPCR amplicon 1 DNAby MethylMiner™ Methylated DNA Enrichment Kit and anti-5mCfrom two different vendors. In parallel, equal moles of anti-5mCantibodymoleculeswerecoupledtoM-280ProteinGDynabeads®andMethylMiner™MBD-biotinmoleculeswerecoupledtoM-280Streptavidin Dynabeads® magnetic beads. After coupling, trip-licate aliquots of 10 µL of antibody-beads were incubated with0.5µg of fragmented, heat-denatured MCF-7 ssDNA and tripli-cate aliquots of 10 µL of MethylMiner™ beads were incubatedwith0.5µgoffragmented,nondenatureddsDNA.Aftermixingat22°Cfor1hour,thesupernatantswererecovered,thebeadswerewashed and then serially eluted with buffers of increasing NaClconcentration.Asafinalstep,allbeadsweretreatedwith20µgofproteinaseKat56°Cfortwohours.TheproteinaseKdigestsuper-natants were collected and extracted with phenol/chloroform/isoamyl alcohol (25:24:1). All fractions were ethanol-precipitated,redissolvedinwater,andassayedwithamplicon1specificprimersbyqPCRusingaSYBR®GreenER™mastermix.Recoveryofampli-fiable DNA was measured relative to a standard curve of inputfragmentedgenomicDNA.Asshown,~100%oftheheavilymeth-ylatedDNAsequencewascapturedby theMethylMiner™beadsand~80%couldbeelutedwith1MNaCl.Incontrast,theanti-5mCantibodyfromtwodifferentvendorscouldonlycapture~10–20%ofthisheavilymethylatedDNAandelutioncouldonlybeachievedwithproteinaseKtreatment.

Figure 15.2. MBD-biotin–based CPG methylated DNA affinity capture. TheMethylMiner™MethylatedDNAEnrichmentKitallows foradiversityofelutionstrategies,dependingonyoupreferredworkflowanddownstreamapplication.Itsupports:1)asingleelutionusingundilutedhigh-saltelutionbuffer;2)asingleelutionusingproteinaseK;3)aseriesofstepwiseelutionswithbufferscontain-ingsuccessivelygreaterNaClconcentrations;or4) stepwise fractionalelutionsfollowedbyafinalproteinaseKtreatment.ThismethodovercomeschallengesassociatedwithdenaturedDNAusingantibody-basedmethods.ThedsDNAismorecompatiblewithdownstreamepigeneticanalysisapplications—includingnext-generationsequencing.

GenomicDNA fragments

Elute CpG-methylateddsDNA as a single fraction

Fractionate CpG-methylateddsDNA with steps of increasing

NaCl concentration

Analyze DNA (+/– bisul�te conversion)by PCR/qPCR, sequencing, microarray, and other assays

2 M NaCl

OR

200 mMNaCl

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

2 MNaCl

CH3

CH3

CH3

CH3

CH 3

CH 3

CH 3

CH 3

Dynabeads®

Streptavidin

Biotin

MBD

CH3

CH3

CH3

CH3CH3

CH3

CH3

CH3

CH3

CH3

Unbou

ndW

ash PK

1,00

0 m

M

600

mM

450

mM

350

mM

200

mM

120%

100%

80%

60%

40%

20%

0%

anti-5mC vendor A

anti-5mC vendor B

MethylMiner™

Figure 15.1. DNA methylation. Amethylgroupaddition to thecytosinecarbon5 incytosine-phosphate-guanine (CpG)andothernucleotidesequencesinhibitsthebindingoftranscriptionfactorstopromoters.

N

C

CN

C C

O

NH2

CH3

Methyl group

Methyl group

G

C

C

G

T

A

G

C

A

T

C

G

T

A

G

C

M M

M M M


15



Enrichment of differentially methylated regions with MethylMiner™ kit fractionation and deep sequencing with the SOLiD™ System

IntroductionDNA methylation is an important epigenetic modification involved in

the remodeling of chromatin structure and, ultimately, the control of

gene expression. Proper control of DNA methylation patterns is essen-

tial for normal embryonic development and tissue differentiation [5],

X-chromosomeinactivation[6],andgeneimprinting[7].Aberrantmeth-

ylation has been linked to many diseases, including cancers [8]. The

modifiedbase5-methylcytosineconstitutesabout1%ofallDNAbases

inmammaliangenomes.Althoughthepresenceof5-hydroxymethylcy-

tosineinbrain[8]andcytosinemethylationatnon-CpGs,namelyCpNpG

inearlyembryogenesis,hasbeenreported[10],mammalianDNAmeth-

ylationisfoundalmostexclusivelyinthesymmetricalCpGdinucleotide.

WhileDNAmethylationinCpG-richpromoterregionscorrelateswithtran-

scriptionalsilencing,itisbecomingclearthatCpGmethylationinregions

of lower CpG density and distal to promoters also correlate with gene

expression [11,12].Thus, itwouldbequiteuseful toeffectivelypartition

denselymethylatedregionsfrommoderatelymethylatedregionstobet-

terunderstandthephysiologicalroleofthisDNAchemicalmodification

TofullyunderstandtheroleofDNAmethylationinnormalanddis-

easestates, it is importanttofirstdeterminegenomicmethylationpat-

terns.Methodsofcharacterizingmethylationaregenerallybasedupon

oneofthreetechniques:bisulfiteconversion,digestionwithmethylation-

sensitive restriction enzymes, and antibody- or 5-methylcytosine bind-

ingprotein–basedpurificationofmethylatedDNA[13].Next-generation

sequencing platforms enable ultrahigh-throughput sequencing, map-

ping,andcountingofshortDNAreads (tags)and, incombinationwith

anyoftheabovemethylationprofilingstrategies,canbeusedforcom-

prehensive,genome-widemappingofmethylationsites.

Methylatedcytosinesconstituteasmallpercentageofallbases

in mammalian genomes. In the human genome, for example, only

about1%of thebasesare5-methylcytosine. It is inmanycasesnei-

ther cost-effective nor necessary to sequence the entire genome

withdeepcoverageinordertointerrogatethegenomicmethylation

patterns. Thus, sensitive, accurate, and versatile tools for the enrich-

mentofmethylatedsequencesfromthegenomearehighlyusefulfor

genome-widestudiesofmethylationpatterns.

Different methods for selective enrichment of methylated

genomicDNAfragmentsareavailable.Thosebaseduponantibodies

aretermedmethylatedDNAimmunoprecipitation(MeDIP)[14].Other

methodsuseproteins thatnaturally recognizemethylatedcytosines

in double-stranded DNA. In the case of the MethylMiner™ system,

thecapturemediumisthemethyl-CpGbindingdomain(MBD)ofthe

human MBD2 protein coupled to superparamagnetic Dynabeads®

M-280 Streptavidin via a biotin linker. Here we describe a genome-

scale study of the patterns of DNA methylation in the MCF-7 breast

cancercell line,usingenrichmentwiththeMethylMiner™systemon

theSOLiD™sequencingsystem.

MethodsComparison of methylated DNA enrichment using the MBD-based

MethylMiner™kittoMeDIP-basedenrichmentidentifiednumerouswork-

flowadvantagestousingtheMethylMiner™system(Figure15.4).First,with

antibody-based enrichment, the fragmented DNA must be denatured

and kept single-stranded during the binding step for efficient capture.

DenaturationoftheDNAisnecessarybecauseallofthevalidatedanti-

bodieshavebeenselectedtobindafullyaccessiblemethylatedcytosine

(5mC)andcannotbind5mCoccurringnaturallyindouble-strandedDNA.

Thisconstraintcanreducebindingefficiency,asfragmentscanreanneal

duringthebindingreaction.Tocompensateforthis,antibody–DNAbind-

ingisoftenperformedforseveralhourstoovernightat4°C.Afterwashing

thebeads,theDNAcanonlyberecoveredafterdigestionwithproteinase

K,usuallywithheat,whichnecessitatesfurtherclean-up,suchasphenol/

chloroformextractionfollowedbyprecipitationwithethanol.Incontrast,

theMethylMiner™systemisfast,convenient,andmoreversatile.TheDNA

remainsdouble-strandedduringthebindingstep,whichonlytakes1hour

atroomtemperature(longertimesat4°Ccanalsobeused).Afterincuba-

tion,theMethylMiner™beadsarewashedandthecapturedmethylated

DNAeluted.Followingadapterligation,theeluteddouble-strandedDNA

fragmentsarereadyfornext-generationsequencing,whichprovideasig-

nificantadvantageoversingle-strandedfragmentsproducedinMeDIP-

based enrichment. The system uses Dynal® superparamagnetic beads,

whichprovidehigh-qualityandefficientcapturekineticsaswellasrapid

andconvenientmagnet-basedbeadcaptureforwashingandchanging

solutions.Batchelutioniseasilyachievedbysimplymixingthebeadswith

ahigh-salt(2MNaCl)buffer[15].Furthermore,sincetheaffinityofMBD2

formethylatedDNAcanbemodulatedbyionicstrength,fractionationof

thecapturedDNAbasedonitsdegreeofmethylationcanbeperformed

withgradedchangesinionicstrength.VaryingdegreesofCpGmethyla-

tiondensitycaninfluencegeneregulationsotheabilitytofractionatethe

genomeaccordingtothedegreeofmethylationisabonusforfunctional

studies. Finally, only ethanol precipitation is needed to obtain concen-

tratedandpurifieddouble-strandedmethylatedDNAthatisreadyforfur-

theranalysis,suchasbyPCRmethodsormicroarrayorhigh-throughput

sequencing(Figure15.4).

ResultsHumanDNAfromthebreastcancer–derivedcelllineMCF-7wasused

tomapthemethylationstatusofCpGsitesonagenomescale.Briefly,

50μgofgenomicDNAwasshearedusingaCovaris™S2non-contact

sonicator(SOLiD™3fragmentlibraryprotocol)togenerateshortran-

domDNAfragmentswithamediansizeof~150bp(Figure15.5A,15.5B).

FragmentedDNAwasthensubjectedtomethylatedDNAenrichment

accordingtotheprotocolsuppliedwiththeMethylMiner™Methylated

DNAEnrichmentKit(Cat.No.ME10025),andtwomethylatedfractions

(500mMand1MNaCleluates)wereisolated[15].Figure15.5Cshows

miR

NA P

rofili

ngIII

137


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Figure 15.4. Comparison of MethylMiner™ and antibody-based MeDIP workflows. (A)Workflowforantibody-basedMeDIP.(B)WorkflowsforMethylMiner™MBDbead–basedenrichmentofCpG-methylatedDNA

Figure 15.5. SOLiD™ 3 fragment library preparation after enrichment and elution using the MethylMiner™ kit. (A) Agarose gel and (B) 2100 Bioanalyzer trace ofshearedhumangenomicDNAsubjectedtoMethylMiner™enrichment.(C)RecoveryofDNAatdifferentstagesofMethylMiner™enrichment.W1–W4aresuccessivewashfractions.0.5a–caresuccessiveelutionswithbuffercontaining500mMNaCl;likewise,1.0a–cand3.5a–caresuccessiveelutionswithbuffercontaining1Mand3.5MNaCl,andPKreferstoresidualDNArecoveredwithafinalproteinaseKtreatment.TheinsetshowsthemassofDNApooledfromtheseiterations.(D)GeloflibraryDNA.

50 b

p ladder

Unfra

gmen

ted h

uman

DNA

Shear

ed fo

r fra

gmen

t libra

ry

350 bp —300 bp —250 bp —200 bp —

150 bp —

100 bp —

50 bp —

15 300200100 1,500500 [bp]

Fluo

resc

ence

uni

ts

0

20

40

60

80

100

1,400

1,200

1,000

800

600

400

200

0W

10.

5b 1.0aW

4W

21.

0c3.

5b PK0.

5a1.

5cW3

1.0b 3.

5a3.

5c

DN

A m

ass

(ng)

DN

A (n

g)

PK3.5 M1.0 M0.5 MWashes0

500

1,000

1,500

2,000Pooled elution fractions

50 b

p ladder

Input

libra

ry

500

mM

libra

ry

1 M

libra

ry

250 bp —

150 bp —

A

B

CD

A B


15



thetypicalelutionprofileofDNAfromtheMethylMiner™beadsfol-

lowing serial salt elution and multiple washes. Greater than 90% of

thecapturedDNAissequentiallyelutedwith500mMand1MNaCl.

TheelutedmassofDNAwas6.9%(3.47μg)ofthetotalmassloaded

(50μg).SubsequentelutionataveryhighNaClconcentration(3.5M)

followedbydigestionwithproteinaseKshowedthatlessthan10%of

thecapturedDNAremainedonthebeadsafterelutionwith1MNaCl.

DNA fractions were then used to construct standard SOLiD™

fragment libraries according to the manufacturer’s instructions

(SOLiD™ Fragment Library Construction Kit). Library DNA was size-

selected either with E-Gel® SizeSelect™ 2% agarose gels or by gel

purification from 2% agarose E-Gel® EX gels (Figure 15.5D). The

MethylMiner™ SOLiD™ libraries were sequenced at 50-base read

length in a 4-well deposition chamber on a SOLiD™ 3 System, and

thesequencedtagsweremappedtothehumanreferencegenome

(hg18) using the SOLiD™ primary and secondary analysis software

package. Each quarter-slide chamber yielded ~20 million uniquely

mappablesequencingreads.

To confirm that MethylMiner™ fractionation was indeed parti-

tioning DNA fragments based on methylation density, the overall

numberofCpGdinucleotideswithinthefragmentsineachfraction-

ationlibrarywascalculatedafterSOLiD™systemsequencing(Figure

15.6).TherewasasignificantlyhigherdensityofCpGdinucleotidesin

theMethylMiner™kit–enrichedfractionscomparedtotheunbound

fraction. Inaddition, the1MNaCleluatehadahigherdensity than

the500mMNaCleluate.Notably,25%oftheunenrichedsequenced

human genomic DNA fragments had no CpGs. In contrast, only

0.65%ofthe500mMNaCl-enrichedsequencesand0.11%ofthe1M

NaCl-enriched sequences contained no CpGs. Despite the fact that

the exact methylation status of the CpG sites was not interrogated

directly,our results suggest that fragmentscontaining twoormore

methylated CpGs per 150 bp were captured by MethylMiner™ kit

fractionationandwereprogressivelyenrichedfrom2-to10-foldwith

increasingdegreesofmethyl-CpGcontent.Overall,ourdatademon-

strate that MethylMiner™ treatment can fractionate DNA based on

CpGdensity[16].

TovalidatetheperformanceofMethylMiner™kit fractionation,

we labeledtheMCF-7DNAfromenrichedandunenrichedfractions

with Alexa Fluor® dyes (BioPrime® Total for FFPE Genomic Labeling

System, Invitrogen)andhybridized themtobothahumanchr21/22

tilingarray (Nimblegen)andahumanCpG island/promoter-focused

array(Agilent)inparallelwithDNAenrichedbytraditionalantibody-

based MeDIP [14]. Summary results for high-intensity microarray

probehitsareshown inFigure15.7.While there isoverlapbetween

thecombinedMethylMiner™kitfractions(500mMand1MNaCl)and

MeDIPdatasets, therearemorethan2,400uniquestronghits from

thecombinedMethylMiner™kit fractionsandonly147uniquearray

hits in theMeDIPsample. In total, therearemorethanfivetimesas

manyhitsoverallandmorethan16timesasmanyuniquehitswith

MethylMiner™kitfractionationascomparedtoMeDIP.

OurresultsindicatethatMethylMiner™fractionationpullsdown

moresequencescontainingCpG-richsitesandsequenceswithinpro-

motersthandoestheantibody-basedapproach.Inadditionto“global”

data demonstrating successful enrichment with MethylMiner™ kit

capture,detailedinformationonlocationsandCpGdensitiescanalso

bedirectlyvisualizedandinspectedwithSOLiD™systemreads.Figure

15.8depictstheuniquelymappableSOLiD™systemsequencingread

locationsanddensitiesonasinglechromosome(chr21).Sequencing

read distributions are depicted for unenriched DNA, successive

MethylMiner™ elution fractions (500 mM and 1 M NaCl), and full

Figure 15.6. CpG dinucleotide density in MethylMiner™ kit–enriched SOLiD™ 3 fragment libraries. Numbers of CpG dinucleotides found withineach 150 bp nuclear fragment sequenced from unenriched DNA andMethylMiner™-enrichedfractions(500mMand1MNaCl)areshown.ThenumberofCpGsperfragmentDNAwasdeterminedbycountingwithin150 bp downstream of uniquely observed read starts. The inset graphshows the 10-fold depletion of CpG-rich fragments from the 500 mMNaClfractionandconcomitant10-foldenrichmentofthesefragmentsinthe1MNaClfraction,bothrelativetounenrichedDNA.

Figure 15.7. Strongly positive features on microarrays: MethylMiner™ kit enrich-ment vs. antibody MeDIP. The Venn diagram shows the strong positivefeatures from chromosomes 21 and 22 (signal intensities >10-fold overbackground) detected on human CpG island/promoter focused arrays.ThenumberofstrongpositivefeaturesforcombinedMethylMiner™frac-tions (red, pooled 500 mM and 1 M NaCl eluates) and antibody MeDIP(blue)areshown.Overlapofstrongfeatures(gray)isalsoshown.

2439 378 147

No. of chr21 and chr22 array probes: 4240

MethylMiner™ kit–enriched

unenriched> 10

anti-5mC MeDIP-enriched

unenriched> 10

0

3 x 106

6 x 106

9 x 106

0 3 6 9 12 15

CpGs per 150 bp fragment

Num

ber

of r

ead

s

Unenriched

500 mM enriched

1 mM enriched

1 x 100

1 x 101

1 x 102

1 x 103

1 x 104

1 x 105

1 x 106

1 x 107

0 4 8 12 16 20 24 28 32

CpGs per 150 bp fragment

Num

ber

of r

ead

s

miR

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Figure 15.8. MethylMiner™ kit enrichment and SOLiD™ system sequencing across chromosome 21. Tracks illustratingsequencingreadsfromunenrichedMCF-7DNA, MethylMiner™ kit–enriched 500 mM and 1 M NaCl elution fractions, and full MethylMiner™ kit enrichment with 2 M NaCl elution are shown.SequencingreaddepthofcoveragefortheenrichedfractionsisshowninredrelativetothemoreuniformlowcoverageobtainedfromunenrichedDNAshowninblue.PositivefeaturesfortheMethylMiner™1MNaCl-enrichedfractiononatilingarrayarealsoshown.LocationsofannotatedgenesandCpGislandsareindicated.Zoomed-inviewsof50kband1kbregionsarediscussedinthetext.


15



MethylMiner™elutionwith2MNaCl;further,tilingmicroarraydatafor

the1MNaClfractionareshown.FortheMethylMiner™kit–enriched

uniquely mapped reads, the read coverage without enrichment is

superimposedontheenrichedreadcoveragetoillustratetherelative

enrichmentobtainedforequivalentamountsofsequencingtimeand

expense;eachsequencingreactioncomprised~20millionuniquely

mappablereadsfromonechamberofa4-wellslide.Theannotated

genesandCpGislandsarealsoshownforreference.Theunenriched

tracksuggeststhattherehasbeenamplificationofchromosome21

DNAinthisvalidatedcancercellline,andtheenrichedtracksindicate

that the distal end of the long arm of the chromosome is densely

methylated. This is not surprising since widespread methylation of

largechromosomalsegmentshasbeenseeninothercancergenome

studiesandsincethevastmajorityofannotatedgenesliewithinthis

regionofchromosome21[17,18].

Atthishighlevel,goodconcordancecanbeseenbetweenthe

datasets,andtheprofilesoftheseriallyeluted1MNaClfractionreads

andthebatch-eluted2MNaClreadsareremarkablysimilar,indicating

good reproducibilityof sequencecaptureandsignificant sequence

overlap between these two different elution schemes. The greater

depthofcoverageseeninthe1MNaClfractionatteststothedecom-

plexingpoweroffractionation.Methylatedsequencesthatresistelu-

tionwith500mMNaClarebetterenrichedinasubsequent1MNaCl

elutionfractionthaninthemorecomplexsamplethatiselutedasa

wholewith2MNaCl.

Foramoredetailedview,theanalysisfocusedona50kbregion

inchromosome21(middleofFigure15.7),andatthislevelofresolu-

tion,abroadpeakofenrichmentthatspanstheneighboringgenes

PFKLandC21orf2canbeseen in theSOLiD™systemdata fromthe

1MNaClelutionfraction.Closer inspectionof the1kbregionthat

encompasses one of the PFKL gene’s intronic CpG islands clearly

demonstrates the enrichment in this fraction (lower portion of

Figure15.8).Theaveragedepthofcoverageislessthan1inboththe

unenrichedand500mMNaClelutionfractions;incontrast,coverage

depthrangesfrom5to15inthe1MNaClelutionfraction.Bisulfite

cloning and sequencing of this CpG island verified that its CpGs

are 94% methylated (Figure 15.8, bottom row). Notably, this CpG

islandfailedtobeidentifiedasmethylatedinourMeDIP-microarray

experiments. To further corroborate the MethylMiner™ SOLiD™

system data with the methylation status of the genomic DNA, we

focusedona200kbregionharboringtheADAMTS1andADAMTS5

genesonchromosome21 (Figure15.9).Thedifferentialenrichment

betweentheMethylMiner™fractionsisevidentatthislocalizedview.

Sequences spanning the body and the upstream promoter region

oftheADAMTS1geneareexclusivelyenrichedinthe500mMNaCl

fraction. With respect to sequences covering the Methylated CpG

Unmethylated CpG CpG island in the promoter of the ADAMTS5

gene, however, there is preferential enrichment in the 1 M NaCl

fraction (Figure 15.8). We further carried out bisulfite cloning and

sequencingacrossaportionoftheCpGislandintheADAMTS5pro-

moter todeterminetheexactmethylationpattern,sincethis locus,

likethePFKLCpGislandshowninFigure15.8,hadalsobeenidenti-

fiedinourmicroarrayexperimentsassignificantlyenriched(>10-fold)

withMethylMiner™treatmentbutnotcapturedbyMeDIP.Asshown

inFigure15.8,thebisulfite-sequencedregionshowsanintermediate

degree (57%)ofCpGmethylation. Inallother loci tested (5 regions

that were similarly detected on microarrays with MethylMiner™ kit

butmissedwithMeDIP),bisulfitesequencinghasconfirmedthepres-

enceof>80%CpGmethylation(datanotshown).Theseresults fur-

therindicatethatMethylMiner™kitenrichmentpermitstheisolation

offractionsofgenomicDNAthatharbordifferingdegreesofmeth-

ylation.Thiscapacitytocapturemoderatelymethylatedregions,and

direct compatibility with SOLiD™ system sequencing, are notable

advantagesoverantibody-basedmethods.

ConclusionsWhenitcomestowhole-genomemethylationanalysis,MethylMiner™

enrichment provides several significant advantages over traditional

antibody-based enrichment. The DNA binding step is rapid, effi-

cient,andmoresensitiveto lowlevelsofCpGmethylationwiththe

biotinylatedMBDprotein inMethylMiner™kit fractionation. Inaddi-

tion, the incorporation of Dynal® superparamagnetic beads facili-

tates effective, high-quality capture kinetics. Furthermore, with the

MethylMiner™kitmethod,genomicDNAcanbefractionatedaccord-

ing to its methylation density. Finally, the capacity to capture and

releasedouble-strandedDNAgreatlyexpedites libraryconstruction

forcurrentgenerationhigh-throughputsequencing.Themultiplex-

ingcapabilitiesofSOLiD™systemsequencingpermitsbarcodingof

numeroussamplesthatcanbesequencedsimultaneously.Moreover,

samples can be further divided into distinct fractions according to

their methylation density with MethylMiner™ kit fractionation. In

summary, MethylMiner™ kit fractionation combined with SOLiD™

systemsequencingisaseamless,easyworkflowthatenablesrobust

enrichment and deep coverage across focal areas for cost-effective

sequencingofmethylatedsequences.

Product Quantity Cat. No.MethylMiner™MethylatedDNAEnrichmentKit 1kit ME10025

miR

NA P

rofili

ngIII

141


www.invitrogen.com

Figure 15.9. Annotated view of a 200 kb region of chromosome 21. TracksofSOLiD™systemreadsfromunenrichedandMethylMiner™kit–enriched500mMand1MNaClfractions.ForMethylMiner™kit–enrichedsequences,thereaddistributionfromanunenrichedsequencedatasetofthesamesizehasbeensubtractedtohighlightregionsofenrichment.LocationsofannotatedgenesandCpGislandsarealsoshown.ResultsofbisulfitesequencingofasmallsegmentinthepromoterCpGislandofADAMTS5arealsoshown.Opencircles:nonmethylatedCpGs;filledcircles:methylatedCpGs.

Methylated CpG

Unmethylated CpG Introduction to DNA methylation

15



Sam

ple

pre

par

atio

n

Genomic DNA isolation and puri�cationPureLink™ Genomic DNA Mini Kit

Bisul�te conversionMethylCode™ Bisul�te Conversion Kit

Amplifying bisul�te-modi�ed DNAPlatinum® Taq Polymerase

Det

ectio

n CloningTOPO® cloning kits for sequencing

Plasmid puri�cation (optional)PureLink™ Quick Plasmid Miniprep Kit

Standard DNA sequencing

Bis

ul�t

e co

nver

sion

Methylation-speci�c PCRPrimers

Figure 15.11. Streamlined MethylCode™ method produces better results. Themost commonly used technique for detecting methylation in specificregionsofDNAisthesodiumbisulfiteconversionmethod.

Locus-specific DNA methylation analysis

Sincethefirstpublishedarticleonthesequencingofbisulfite-convertedDNAin1992[19],thebisulfiteconversionmethodhasbecomeapre-

ferredtoolformethylationanalysis.Inthismethod,DNAisdenaturedandtreatedwithsodiumbisulfite,causingunmethylatedcytosinestobe

convertedtouracilswhilemethylatedcytosinesremainunchanged[19].ConvertedDNAcanbePCR-amplifiedandanalyzedbyDNAsequencing

orrestrictionendonucleasedigestionandthemethylationpatternsdeterminedbycomparisontountreatedDNA(Figure15.10,Figure15.11).

Genome-wide methylation

Methylationofcytosines inmammaliangenomicDNA isnowa recognizedepigeneticphenomenonwithwide-ranging implications inbasic

generegulationandmedicalresearch.Inparticular,changesinDNAmethylation,includinggenome-widelossesandhypermethylationofpro-

moterregions,arestronglycorrelatedwiththeonsetandprogressionofcancer.Onecommonstrategyformonitoringthemethylationstatusof

thegenome,andparticularlociwithinit,involvesfragmentingtheDNA,andthencapturingthemethylatedfragmentswithananti–5-methyl

cytosineantibody.TheMeDIP(methylatedDNAimmunoprecipitation)assayhasbeenusedinconjunctionwithbisulfitesequencingtodetermine

theprecisedistributionofmethylatedcytosinesinthreehumanchromosomesandintheArabidopsisgenome.Variationsonthisassay(MCIPand

MIRA)havebeendescribedthatutilizemethyl-bindingdomain(MBD-family)proteinsinlieuofanantibody.

GenomicDNAisolationandpurificationformethylationanalysis

SuccessfulresultsbeginwitheffectivegenomicDNAisolationandpurification.InvitrogenoffersseveraloptionsforgenomicDNAisolation:

→ PureLink™reagents—silicaspin-columntechnology

→ ChargeSwitch®-magneticbeadbasedpurification,amenabletohigh-throughputisolation

Visitwww.invitrogen.com/oligosforinformationonprimerdesignsoftwaretohelpwithlow-complexityDNA.

Original DNA with methylated CmpG

DNA sequencing after CT conversion

151413121110987654321

CCGTCACTCGCmGTTG

TTGTTATTTGCGTTG

Figure 15.10. Complete conversion of unmethylated cytosines into uracils after bisulfite treatment. DNAwithmethylatedCpG(position5)wasprocessedusingtheMethylCode™BisulfiteConversionKit.TherecoveredDNAwasamplifiedbyPCR,cloned,andsequenced.Followingbisulfitetreatment,themethylatedcytosineatposition5remainedintactwhiletheunmeth-ylatedcytosines(positions7,9,11,14,and15)wereconvertedintouracilsanddetectedasthyminesfollowingPCR.

miR

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PureLink™ reagents—reliable isolation of genomic DNA

PureLink™genomicDNAisolationreagentsenablehigh-yield,high-purityDNAextractionsfromawidevarietyofsampletypes,includingblood,

tissue,cells,bacteria,swabs,andbloodspots,inafamiliarsilicaspin-columnorsemi-skirted96-wellplateformat.WithGenomicDNAKitsallow

youto:

→ ObtainhighergDNAyieldsandpurity,withoptimizedspin-columnmembraneandbufferformulations

→ Savetimeandmoney—usewithalargerangeofsamplesizes(e.g.,100μLto1mLblood);96-wellversionsdonotrequirespecialequipment

→ Increaseflexibility—onekitworkswithavarietyofsampletypes

Product Quantity Cat. No.PureLink™GenomicDNAMiniKit 10preps K1820-00

PureLink™GenomicDNAMiniKit 50preps K1820-01

PureLink™GenomicDNAMiniKit 250preps K1820-02

PureLink™96GenomicDNAKit 4x96preps K1821-04

PureLink™GenomicDigestionBuffer 70mL K1823-01

PureLink™GenomicLysis/BindingBuffer 80mL K1823-02

PureLink™GenomicWashBufferI 100mL K1823-03

PureLink™GenomicWashBuffer2 75mL K1823-04

PureLink™GenomicElutionBuffer 160mL K1823-05

PureLink™Pro96GenomicDNAPurificationKit 4x96preps K1821-04A

EveryPrep™UniversalVacuumManifold 1each K2111-01

PureLink™GenomicPlantDNAPurificationKit 50preps K1830-01


15



Figure 15.12. Effects of bisulfite treatment. Unmethylatedcytosinesareconvertedtouracils,whilemethylatedcytosinesremainunchanged.

Original DNA with methylated CpG: G T T G Cm G C T C A C T G C C Same DNA after bisul�te treatement: G T T G Cm G U T U A U T G U U

Figure 15.13. Streamlined MethylCode™ bisulfite conversion protocol is complete in just 3 hr. A.SampleDNAistemperature-denaturedandconverted,andtheconvertedDNAisprecipitatedusinganin-columndesulfonationtechnologyandtheneluted.B.TheDNAcanthenbePCRamplified,cloned,sequenced,andanalyzedformethylationpatterns.

TOPO

TOPO

TOPO®

cloningvector

PCR ProductCACC

Design conversion-speci�c primers and PCR amplify

Clone PCR product for sequencing

Sequence and analyze methylation status

Elute

Mix sample DNA and CT conversion reagent

Incubate at 98°C for 10 min, then 64°C for 2.5 hr

Clean up and desulfonate

PCR-ready DNA

A

B

Product Quantity Cat. No.MethylCode™BisulfiteConversionKit 50rxns MECOV-50

MethylCode™ bisulfite treatment—fast and complete DNA conversion

ThemostcommonlyusedtechniqueforDNAmethylationanalysisconsistsoftreatingDNAwithsodiumbisulfite,whichcausesunmethylated

cytosines to be converted to uracils while methylated cytosines remain unchanged (Figure 15.12). The MethylCode™ Bisulfite Conversion Kit

improvesuponthisapproach, increasingconversionefficiency,while reducingthenumberofstepsandoverall timerequired for results.The

streamlinedprotocol(Figure15.13)integratestheDNAdenaturationandbisulfiteconversionprocessesintooneconvenientstep,replacingchemi-

caldenaturationusingsodiumhydroxidewithasimpletemperaturedenaturation.Inaddition,cumbersomepostconversionDNAprecipitation

stepsareeliminatedbyaninnovativein-columndesulfonationtechnology.ThisminimizestemplatedegradationandDNAlosssothataslittle

as500pgofstartingmaterialisrequired.RecoveredDNAisreadyforPCRamplificationanddownstreamanalysisapplicationsincludingrestric-

tionendonucleasedigestion,sequencing,andmicroarrays.Completeconversionofunmethylatedcytosines,aswellasDNApurification,canbe

achievedinlessthan3hours,comparedtotraditionalmethodsthatrequireovernightincubation.TheMethylCode™BisulfiteConversionKitoffers

manybenefits:

→ Completeconversionofunmethylatedcytosines—allowsyoutoeliminatefalsepositives

→ MinimaltemplatedegradationandDNAlossduringtreatmentandclean-up

→ Highyieldofbisulfite-convertedDNA

→ Lesstimeatthebench—integratedone-stepDNAdenaturationandbisulfiteconversioncompletedin3hr

→ Simplifiedprocedure—enableseliminationofcumbersomeDNAprecipitationstepsusingnovelin-columndesulfonation

miR

NA P

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www.invitrogen.com

Detection of bisulfite-modified DNA

Detectionofbisulfite-modifiedDNAcanbefacilitatedthroughconventionalPCR,methylation-specificPCR,andbisulfitegenomicsequencing

afterPCRamplification,withorwithoutcloning.

High-fidelity reagents for amplifying bisulfite-treated DNA and methylation-specific PCR (MSP)*Followingbisulfiteconversion,themodifiedDNAtemplateisdistinguishablefromtheoriginaltemplateatmethylatedcytosines.Thisresultsina

poolofDNAfragmentswithalterednucleotidesequencesduetodifferentialmethylationstatus.Themethylation-specificPCRreactioninvolves

amplifyingthealterednucleotidesequencesusingmethylation-specificprimers fortheCpGislandsof interest (Figure15.14). Invitrogen’sPCR

productsaretailoredtoaddressthepersistentchallengesassociatedwithamplifyingbisulfite-modifiedDNA—suchasasmallinitialsampleof

highlyfragmentedDNA.NotallpolymerasesareeffectiveatamplifyingconvertedDNA,duetovariationinspecificityandfidelityoftheparticular

enzymesused.WehavevalidatedtheuseofPlatinum®TaqDNApolymerasesforamplificationofbisulfite-convertedDNA.UsingthePlatinum®

PCRkits,youcanexpect:

→ Faithfulamplificationbasedonreliableandconsistentsingle-nucleotidediscrimination

→ Highsensitivityandhighyieldofbisulfite-convertedDNA

→ Room-temperaturereactionassembly

*Methylation-specificPCRmaybecoveredbyoneormoreU.S.patents—nos.5,786,146;6,017,704;6,200,756,and6,265,171,andpatentsbasedonforeigncounterpartapplications.Nolicenseorrightsunderthesepatentstoperformmethylation-specificPCRisconveyedexpresslyorbyimplicationtothepurchaseofInvitrogen’sproduct.UsersofInvitrogenproductssubjecttothislabellicenseshoulddeterminewhethertheyhavealltheappropriatelicensesinplace.Further,nowarrantyisprovidedthattheuseoftheseproductswillnotinfringeonthepatentsreferredtoabove.

Product Quantity Cat. No.TaqDNAPolymerase,native 100units 18038-018

TaqDNAPolymerase,native 500units 18038-042

TaqDNAPolymerase,recombinant 100units 10342-053

TaqDNAPolymerase,recombinant 500units 10342-020

Platinum®TaqDNAPolymerase 100rxns 10966-018



Estrogen receptor α (ERα) Unmethylated CpG Methylated CpG

MCF-7

MDA-MB-231

CpG 1 CpG 2 CpG 3 CpG 4 CpG 5 CpG 6 CpG 7 CpG 8 CpG 9 CpG 10 CpG 11 CpG 12 CpG 13 CpG 14 CpG 15 CpG 16

Figure 15.14. Methylation patterns of ERα as analyzed using the MethylCode™ kit. GenomicDNAfromMCF-7andMDA-MB-231celllineswasisolatedandtreatedwiththeMethylCode™BisulfiteConversionKit.Bisulfite-convertedDNAwasthenusedastemplateinPCRtoamplifyaportionontheestrogenreceptoralpha(ERα)gene.Next,thePCRproductwasclonedintoasequencingvectorandsequenced.Finally,thesequenceswereanalyzedtoshowadifferentialmethylationpatternoftheERαgenebetweentheMDA-MB-231andMCF-7celllines(locus—X036356450bpmRNAlinearPRI11-JUN-2003;definition—HomosapiensmRNAforestrogenreceptor;accession—X03635M11457;version—X03635.1GI:31233).


15



Simplify your preparation for bisulfite genomic sequencing

CombineeffectiveplasmidDNApurificationwithquickandefficientcloning.ThePureLink™QuickPlasmidMiniprepKitprovidesefficientand

reliablepurificationofplasmidDNAforcloningwithTOPO®technology.TOPO®technologypresentsthefastest,mostefficientwaytoclonePCR

productsforsequencing.ThekeytoTOPO®cloningistheenzymeDNAtopoisomerase I,whichfunctionsasbotharestrictionenzymeanda

ligasethatcleavesandrejoinsDNAduringreplication.Toharnessthereligatingactivityoftopoisomerase,weprovideover30linearizedvectors

withtopoisomeraseIcovalentlyboundtoeach3’phosphate.Typically,afteronly5minutesatroomtemperature,ligationofDNAsequenceswith

compatibleendsligationsiscompleteandyourDNAisreadyfortransformationintoE. coli.

Top-rated sequencing vectors

TheTOPO®TACloningKitsforSequencingcontainavectorwithaminimizedmultiplecloningsitethatpositionstheT7andT3primingsitesonly

33bpawayfromthePCRproductinsertionsite.Thisallowsyoutosequencemoreofyourinsertandlessofthevector,savingyoutimeandmoney.

ItisdesignedforfastandefficientresultsfromTaq-amplifiedPCRproducts.

Product Quantity Cat. No.TOPO®TACloning®KitforSequencingwithOneShot®MAXEfficiency™DH5α-T1RE. coli 1kit K4595-01

TOPO®TACloning®KitforSequencingwithOneShot®MAXEfficiency™DH5α-T1RE. coli 40rxns K4595-40

TOPO®TACloning®KitforSequencing(withpCR4-TOPO®)withOneShot®TOP10ChemicallyCompetentE. coliandPureLink™QuickPlasmidMiniprepKit

20preps K4575-02

TOPO®TACloning®KitforSequencingwithOneShot®TOP10ChemicallyCompetentE. coli 10rxns K4575-J10

TOPO®TACloning®KitforSequencingwithOneShot®TOP10Electrocomp™E. coli 20rxns K4580-01

TOPO®TACloning®KitforSequencingwithOneShot®TOP10Electrocomp™E. coli 40rxns K4580-40

TOPO®TACloning®KitforSequencing 40rxns K4575-40

TOPO®TACloning®KitforSequencing 20rxns K4575-01

TOPO®TACloning®forSequencingwithOneShot®Mach1™T1Phage-ResistantChemicallyCompetentE. coli 20rxns K4530-20

TOPO®TACloning®KitDualPromoter(withpCRII-TOPO®vector)withOneShot®Mach1™T1Phage-ResistantChemicallyCompetentE. coli

20rxns K4610-20

TOPO®XLPCRCloningKitwithOneShot®Mach1™T1Phage-ResistantChemicallyCompetentE. coli 20rxns K7030-20

TOPO®TACloning®Kit(withpCR2.1-TOPO®vector)withOneShot®Mach1™T1Phage-ResistantChemicallyCompetentE. coli 20rxns K4510-20

Chapter references

1. AdamsRL(1995)Bioessays17:139–145.

2. OzanneSEetal.(2007)Nat Clin Pract Endocrinol Metab3:539–546.

3. DolinoyDCetal.(2007)Reprod Toxicol23:297–307.

4. VermaMetal.(2006)Crit Rev Oncol Hematol60:9–18.

5. MorganHD,SantosF,GreenKetal.(2005)Hum Mol Genet14:R47–R58.

6. ReikW,LewisA(2005)Nat Rev Genet6(5):403–410.

7. FeinbergAP(2007)Nature447(7143):433–440.

8. JonesPA,BaylinSB(2007)Cell128(4):683–692.

9. KriaucionisS,HeintzN(2009)Science324(5929):929–930.

10. RamsahoyeBH,BiniszkiewiczD,LykoFetal.(2000)Proc Natl Acad Sci U S A97(10):5237–5242.

11. SchmidlC,KlugM,BoeldTJetal.(2009)Genome Res19(7):1165–1174.

12. IrizarryRA,Ladd-AcostaC,WenBetal.(2009)Nat Genet41(2):178–186.

13. ZilbermanD,HenikoffS(2007)Development134(22):3959–3965.

14. WeberM,DaviesJJ,WittigDetal.(2005)Nat Genet37(8):853–862.

15. MethylMiner™manual;followlinksfrombottomofthefollowingpage:www.invitrogen.com/methylminer.

16. MethylMiner™forMethylatedDNAEnrichmentwebinar:http://invitrogen.cnpg.com/Video/flatFiles/1080/index.aspx.

17. ClarkS(2007)Hum Mol Genet16:R88–R95.

18. GardinerK,HorisbergerM,KrausJetal.(1990)EMBO J9(1):25–34.

19. FrommerMetal.(1992)Proc Natl Acad Sci U S A89:1827–1831.

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15

Section IV—Histone Modifications and Chromatin Remodeling

Chapter 16—Histone modifications and chromatin remodeling . . . . . . . . 150


Section IV Histone Modifications and Chromatin Remodeling

CHAPTER 16

Histone modifications and chromatin remodelingDNAiscompactedandorganizedwithinchromatin inthenucleiofeukaryoticcells.Asthefundamentalsubunitsofchromatin,nucleosomes

formachainofellipsoidalbeadsofhistoneproteinsaroundwhichtheDNAiswound.Fourhistonetypes(H2A,H2B,H3,andH4)formtheoctamer

centerofthenucleosome,with147nucleotidepairswrapped1¾turnsaroundthecenter[1].Eachhistonehasanamino-terminaltailregioncon-

sistingof25–40aminoacidresiduesthatprotrudebeyondthenucleosomesurface.H1histoneplaysaroleinlinkingthenucleosomestructures

togethertocondensethechromatin.

Chromatinexistsinmanyconfigurationsandundergoesdynamicstructuralchanges.Theserangefromlocalchangesnecessaryfortran-

scriptional regulation toglobalchangesnecessary forchromosomesegregation.Severalepigeneticmechanisms introducevariation tochro-

matin structure, including covalent histone modifications, histone variant composition, DNA methylation, and non-coding RNA. Changes to

thechromatinstructure,calledchromatinremodeling,canfacilitategenetranscriptionbylooseningthehistone-DNAcomplex,allowingother

proteinssuchastranscriptionfactorstoaccesstheDNA.Alternatively,chromatinremodelingwherehistonesassumeamoreclosedconformation,

blockstranscriptionfactoraccesstotheDNA,resultinginlossofgeneexpression.Relativelylittleisknownabouthowremodelingfactorschange

nucleosomestructureandhowdifferentfactorsworktogethertopromotechromatinremodeling.

Chromatinremodelingistypicallyinitiatedbyposttranslationalmodificationoftheaminoacidsthatmakeupthehistoneproteins,aswell

asthroughmethylationofneighboringDNA(Figure16.1).

ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPH Histone H3 135 aa2 4 9 10 14 1718 23 26 27 28 36 79

S F R F K G G K G L G K G G A K R H R K V L R D N I Q G I T K PA I R R L A R Histone H4 102 aa1 3 5 8 12 16 20

SGRGKQGGKARAKAKSRSSRAGLQFPVGRVHRLLRKGNY Histone H2A 129 aa1 5 9 119

PEPAKSAPAPKKGSKKAVTKAQKKDSKKRKRSRKESYSV Histone H2B 125 aa5 12 14 120

Acetylation

Methylation(arginine)

Methylation(active lysine)

Methylation(repressive lysine)

Ubiquitination

Phosphorylation

Figure 16.1. Histone modifications and variants. Posttranslationalmodificationstohistoneamino-terminaltailsincludeacetylation(lysineandarginine),phos-phorylation(serineandthreonine),ubiquitination(lysine),andsumoylation,whichareassociatedwithawidevarietyofbiologicalprocesses includingtranscriptionalactivationandrepression,mitosis,andapoptoticregulation.

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Investigating histone modifications and chromatin remodeling

Histonesaresubjecttoawidevarietyofposttranslationalmodifications,includingbutnotlimitedtolysineacetylation,lysineandargininemeth-

ylation,serinephosphorylation,andlysineubiquitination.Thesemodificationsoccurprimarilywithinthehistones’amino-terminaltailsandare

thoughttoaffectchromosomefunction.Posttranslationalmodificationsofhistonescreateanepigeneticmechanismforregulatingavarietyof

normalanddisease-relatedprocesses.Currentresearchhasfocusedoninvestigatingtheroleofepigenetics intranscription,cellproliferation,

differentiation,senescence,apoptosis,andtheDNAdamageresponse.

MAGnify™ ChIP

Todate,themostwidelyusedandpowerfulmethodtoidentifyregionsofthegenomeassociatedwithspecificproteinsisthechromatinimmu-

noprecipitation(ChIP)assay.TheChIPassayhasbeenwidelyusedtostudybothhistoneandnonhistoneproteins,suchastranscriptionfactors,

withinthecontextofthecell.Antibodiesthatrecognizeaproteinofinterestareusedtodeterminetherelativeassociationofthatantigeninthe

contextofchromatinatoneormorelociinthegenome.

IntheChIPassay,chromatinisshearedandimmunoprecipitatedwithahighlyspecificantibodydirectedagainstacomponentofthechro-

matincomplex.BecausetranscriptionfactorsandotherDNA-bindingproteinshaveaweakeraffinityforchromatinthanhistones,acrosslinking

stepisincorporatedbeforeimmunprecipitationtoavoiddissociationofnonhistoneproteinsfromthechromatin-bindingsite.

Histone modifications and chrom

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MAGnify™ Chromatin Immunoprecipitation System

ThiskitfacilitatesafasterandmorereproduciblesolutionforChromatinImmunoprecipitation(ChIP)andincludesallreagentsneededtoperform

ChIPwithanantibodyofinterest.TheMAGnify™ChromatinImmunoprecipitationSystemwillallowyouto:

→ ReduceoverallChIPprotocoltimebyoneday(Table16.1)

→ ReduceinputcellnumberperChIPexperiment(10,000–300,000cellsrequired)(Figures16.2,16.3)

→ DecreasebackgroundcausedbynonspecificbindingthroughtheuseofDynabeads®

→ ImprovereproducibilityduetooptimizedmagneticDNApurification(avoidcolumnsandphenol/chloroformsteps)(Figure16.4)

→ Increase confidence in results due to optimized and reproducible components and antibodies that have been qualified for chromatin

immunoprecipitation

→ Easilyincreasethroughputwithsmallvolumes,magneticprotocol,andmagnetcompatiblewithmultichannelpipetting

→ ReduceexperimentalerrorwithDynabeads®ProteinA/GMix—worrylessaboutantibodycompatibility

The ChIP-ready DNA is ready for downstream analysis for most methods, including PCR/qPCR-based assays, bisuflite conversion followed by

amplification,cloning,sequencing,directsequencing,librarypreparationforhigh-throughputsequencing,andsampleprepforDNAmicroarray

analysis.ThiskitisuniqueinitsutilityforpermittingresearcherstotakeadvantageoflowerstartingcellnumbersforChIP,thuspreservingpre-

cioussamplessuchasprimarycells,stemcells,andbiopsies.AnotherkeyaspectforsuccessfulChIPanalysisisantibodiesthatrecognizethetarget

proteininthecontextofchromatin.WehavedevelopedanextensivecatalogofCHiP-testedantibodiestoexpeditesuccess.

Table 16.1. Comparison to a conventional ChIP protocol.

Workflow step MAGnify™ ChIP timeline Conventional ChIP timeline

Preclearing N/A 1–2hr

Antibody/chromatinincubation 2hr Overnight

Beadpulldown 1hr 2hr

Washes 30min(2buffers) 1–3hr(4buffers)

Reversecrosslinking

1.5hr

Overnight

ProteinaseKdigestion 2hr

DNAelutionfrombeads 15–30min

DNApurification 2hr–overnight

Average time 5 hr 36–48 hr

Figure 16.2. Titration of cell number with MAGnify™ ChIP. Sheared chro-matinfromMCF7cellswaspreparedfrom106cells in50μLlysisbuf-fer and diluted to indicated number of cells per ChIP according tothe MAGnify™ ChIP protocol. 1 μg of antibody (IgG included in kit:H3-K27Me3,Cat.No.49-1014;H3-K9Ac,Cat.No.49-1009)wereusedforeachChIPexperiment.1%ofthesonicatedchromatinwassetasideasinputcontrol.OptimizedqPCRprimerswereusedtoamplifytheSAT2locus(Cat.No.49-2026).Dataaregraphedaspercentinput.

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H3-K27Me3IgG RNA Pol II H3-K9AcH3-K27Me3IgG RNA Pol II

0.5

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1.5

2.5

3.5

4.5

2.0

3.0

4.0

RARb1 promoter ER-α promoter

Figure 16.3. 10,000 cell input with MAGnify™ ChIP. ShearedchromatinfromMCF7orMDA-MB231cellswaspreparedfrom106cells in50μLlysisbufferanddilutedto10,000cellsperChIP,accordingtotheMAGnify™ChIPprotocol.1μgofantibody(IgGincludedinkit:H3-K27Me3,Cat.No.49-1014;H3-K9Ac,Cat.No.49-1009)or3μLRNAPolII(Cat.No.49-1033)wereusedforeachChIPexperiment.1%ofthesonicatedchromatinwassetasideasinputcontrol.OptimizedqPCRprimerswereusedtoamplifytodifferentloci:RARb1promoter(Cat.No.49-2027)orER-αpromoter(Cat.No.49-2028).Dataaregraphedaspercentinput.

Figure 16.4. MAGnify™ ChIP vs. conventional protocols. Shearedchromatinfrom293Gtcellswaspreparedand50,000cellsusedforChIP(MAGnify™ChIP)and106cellsperChIPusedfortheconventionalChIPprotocol.1μgofantibody(IgGincludedinkit:H3-K9Me3,Cat.No.49-1008)wasusedforeachChIPexperi-ment.1%ofthesonicatedchromatinwassetasideas inputcontrol.OptimizedqPCRprimerswereusedtoamplifytheSAT2 locus(Cat.No.49-2026),aknowntargetofheterochromatin-associatedH3-K9Me3.Keyelementsofaconventionalprotocoluseanovernightimmunoprecipitationstepofantibodyandchromatin,followedbyenrichmentwithProteinAsepharosebeadsandextensivewasheswithbufferscontainingvariablesaltconcentrations.Reversecrosslinkingwasdoneovernightat65°CandDNApurificationperformedbyphenol/chloroformextraction.

Product Quantity Cat. No.MAGnify™ChromatinImmunoprecipitationSystem 1kit 49-2024

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Antibodies qualified for chromatin immunoprecipitation

AntibodiesareusedinChIPtocapturethecrosslinkedDNA–proteincomplex.PerformingasuccessfulChIPassayrequiresthattheantibodyhas

highaffinityforthefixedproteinthatisboundtothechromatincomplex.BecauseantibodiesarecrucialforasuccessfulChIPexperiment,itis

importanttoselectanantibodythathashighaffinityfortheproteinorproteinsofinterest.Theaffinityforparticularepitopescandifferbetween

differentantibodiesandantibodypreparations.Differencesinaffinityoftheantibodywillaffectthestrengthofassociationwiththechromatin

complex,andtheresultingsignallevels.Further,someantibodiesmaybemoresensitivetoinhibitoryfactorspresentinthechromatinsample,

resultinginadecreaseinbindingefficiencywithincreasinginput.

Unfortunately,thesuccessfuluseofaspecificantibodyinotherapplications(i.e.,westernblotting)maynotguaranteesuccessinChIPanaly-

sis.Toensuresuccess,wehavequalifiedanumberofantibodiesdirectedagainsthistones,aswellasotherchromatinassociatedproteinsforuse

inChIPanalysis.PleasenotethatwecarryanextensiveselectionofantibodiesthathavebeenprequalifiedforuseinChiPanalysis.

NOTE: Please visit our antibodies page at www.invitrogen.com/chipantibody for an up-to-date listing of ChIP-qualified antibodies.

DynaMag™-PCR

TheMAGnify™ChIPsystemusesanovelmagnetthatiscompatiblewith0.2mLPCRstriptubes(Figure16.5).ThenewDynaMag™-PCRmagnet

isoptimizedforefficientmagneticseparationofsmallsamplevolumesusingDynabeads®magneticbeads.Thismagnethasbeendesignedfor

applicationswithreducedstartingcellnumbersandvolumesusedinimmunoprecipitationandwashingsteps.Thenovelmagneticseparator

allowsyoutoachieveresultsquicklyandeasilywhileofferingthefreedomtosimultaneouslyperformmultipleChIPassaysinPCRtubeswitha

multichannelpipettor.

→ Optimalworkingvolume:upto200µL

→ Holdsuptosixteen0.2mLPCRtubes(individualorstriptubes)

Product Quantity Cat. No.DynaMag™-PCR 1each 49-2025

Figure 16.5. DynaMag™-PCR magnet.

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Quantitative PCR (qPCR) of ChIP-ready DNA

Goodprimerdesigniscriticalforhigh-qualityChIPdata.Ingeneral,primersshouldbe20to30baseslongwithaTmbetween55°and60°C.Most

primersdonotrequirepurificationorspecialtreatmentpriortoPCR.Primersshouldbedesignedsothatamplificationtargetsare75to350bpin

length,asamplificationefficiencyisreducedsubstantionallywithincreasinglength.Ingeneral,afinalprimerconcentrationof1μMworkswellfor

mostprimersets,butinsomeinstances,improvedproductspecificitymaybeobtainedbyloweringthefinalprimerconcentration5-to10-fold.

High-qualityprimerpairsshouldresult in~1.9-foldamplification/cycle(thiscanbedeterminedfromquantitativeanalysisofrawfluores-

cencedataforeachcycle,whichisgenerallyavailableoncommercialinstruments).AmplifiedmaterialatthecompletionofthePCRshouldcon-

tainonlyoneproduct(asassayedonhigh-percentageagaroseorpolyacrylamidegels).Specificityinformationcanalsobeobtainedbyrunning

dissociationcurvesonreactionsfollowingtheconclusionoftheqPCRrun.

Ingeneral,individualsamplesshouldberunintriplicate.Foreachprimerpairexamined,theinputDNAsamplesshouldberunalongsidethe

immunoprecipitatedsamples.Amplificationefficienciesamongdifferentprimerpairsvaryslightlyonaper-cyclebasis,andtheseslightvariations

inefficiencycantranslateintosubstantiallydifferentamountsofamplifiedmaterialinthecyclerangeusedforanalysis.Precisequantitationof

relativebindingcannotbeaccuratelyperformedwithoutaprimerpair–specificinputsignal.

WehavevalidatedChIPprimerpairsforvariouspromoterregionsforvalidationofChIP-qPCR.Primerswerevalidatedbytesting5-pointdilu-

tionsofinputDNAfromMCF7andPC3cells,withprimerefficiencyandslopeassessedforeachpair.

Product Quantity Cat. No. MAGnify™SAT2Primers 100reactions 49-2026

MAGnify™RARβ1Primers 100reactions 49-2027

MAGnify™ERαPrimers 100reactions 49-2028

MAGnify™c-FosPrimers 100reactions 49-2029

Chapter reference

1.LugerKetal.(1997)Nature389:251–260.


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Aacetylation ....................................................................................151adenocarcinoma................................................................................121adenoviralRNAivectors ............................................................................ 18adenovirus ...................................................................................17,22AlexaFluor®dyes ................................................................... 21–22,46,118,120,138Ambion®Pre-miR™miRNAPrecursors................................................................109,111Ambion®Silencer®SelectsiRNA. See Silencer®SelectsiRNAAnti-miR™mimicsandinhibitors ......................................................................109Anti-miR™miRNAInhibitors ...................................................................... 109–111

Bbisulfiteconversion..................................................................... 136,142,144–145bisulfitesequencing ........................................................................ 134,140–142BLOCK-iT™AdenoviralRNAiExpressionSystem ................................................... 18,54,68–69,71,72BLOCK-iT™AlexaFluor®RedFluorescentControl ....................................................... 7,72,79–81BLOCK-iT™fluorescentcontrols ........................................................................ 21BLOCK-iT™FluorescentOligos.............................................................. 12,22,75,79–81,85BLOCK-iT™HiPerform™LentiviralPolIImiRRNAiExpressionSystem............................................. 58,60,64BLOCK-iT™InducibleH1LentiviralRNAiSystem..........................................................68–69,72BLOCK-iT™InducibleH1RNAiEntryVectorKit............................................................... 54BLOCK-iT™LentiviralPolIImiRRNAiExpressionSystem.....................................18,54–55,57–58,62,64,68–69,72BLOCK-iT™LentiviralRNAiSystems.................................................................. 18,70,72BLOCK-iT™miRRNAiSelect......................................................................57,64–65BLOCK-iT™PolIImiRRNAi.........................................................................93,95BLOCK-iT™PolIImiRRNAiexpressionvectors .................................................53–54,56–57,61–65,75BLOCK-iT™PolIImiRRNAivectors.................................................................... 92–93BLOCK-iT™PolIImiRValidatedmiRNAControlVectors.......................................................79,85BLOCK-iT™PolIIValidatedmiRNAControlVectors............................................................ 64BLOCK-iT™RNAiAdenoviralSystem...................................................................18,68BLOCK-iT™RNAiBasicControlKit....................................................................... 76BLOCK-iT™RNAiDesigner..................................................................... 8,45,64–66BLOCK-iT™RNAiEntryVectorKits....................................................................... 66BLOCK-iT™RNAiExpress............................................................................ 28BLOCK-iT™RNAiExpresssearchengine ................................................................... 64BLOCK-iT™RNAivectors ............................................................................ 53BLOCK-iT™shRNAvectors....................................................................53–54,92,94BLOCK-iT™siRNA.......................................................................... 22–23,48–49BLOCK-iT™TransfectionKit ....................................................................... 75,79,81BLOCK-iT™TransfectionOptimizationKit ................................................................79,84BLOCK-iT™U6RNAiEntryVectors ....................................................................54,66

Ccancer.........................................................6,30,32,100,125,126,129–130,134,136,140,142cellcycleregulation..............................................................................134Cells-to-Ct™Kits .............................................................................. 90,113chromatinimmunoprecipitation(ChIP) ............................................................... 151–154chromatinremodeling......................................................................... 150–155cloning

TOPO®technology...........................................................................146CpGdinucleotides ................................................................... 134,136,138–141,145customsiRNAlibraries ............................................................................. 51

157

Index

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DDicer .......................................................................................4–6DNAmethylation.......................................................................134,142,144,150DNApolymerases...............................................................................145dsRNA......................................................................................15,40dyeswap. See loopdesign/dyeswapDynaMag®-PCR .................................................................................154

Eelectroporation .................................................................................. 15embryonicdevelopment........................................................................134,136epigenetics. See DNAmethylation,chromatinremodeling,noncodingRNAs

FFastPrep®-24Instrument............................................................................ 24

GGateway®technology ................................................................... 55,62,64,66,93–94geneimprinting.............................................................................134,136genomicDNAisolation ......................................................................... 142–143

HHistoGrip™ ..................................................................................... 24histonemodifications .............................................................................150

Iimmunoprecipitation.......................................................................... 152–153InduciblePolIImiRRNAiExpressionVectorKit.............................................................. 62inducibleRNAi.................................................................... 7,9,10,63,67,72,93,96interferonresponses............................................................................... 40in vivoRNAi...........................................................................19–28,44,46,92

Llabeling......................................................................................118Latinsquaresnormalization.........................................................................122lentiviraldelivery................................................................................. 22LentiviralPolIImiRRNAiExpressionSystem. See BLOCK-iT™LentiviralPolIImiRRNAiExpressionSystemlentiviralRNAidelivery......................................................................17–18,68,70Lipofectamine®2000TransfectionReagent................................................7–8,12,60,72,75,81,84–85Lipofectamine®LTXReagent ........................................................................ 8,72Lipofectamine®RNAiMAXTransfectionReagent.............................................. 7,10–13,15,72–75,80–81locationanalysis................................................................................128loopdesign/dyeswap............................................................................122

MMAGnify™ChromatinImmunoprecipitation(ChIP)System .................................................. 152–154MeDIP.................................................................................138,140,142.

See also methylatedDNAimmunoprecipitationMegaplex™primers...........................................................................104,106methylatedDNAimmunoprecipitation ............................................................ 136–138,142methylation................................................................................ 144–145methylation-specificPCR...........................................................................145MethylCode™BisulfiteConversionKit ............................................................. 142,144–145

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methyl-CpGbindingdomain. See DNAmethylationMethylMiner™MethylatedDNAEnrichmentKit ................................................... 134–135,138,140microarrays......................................................91,103,114,118,120,122,124–127,138,140,144miRNA............................................................................9,34,57–58,98–114

amplification ............................................................................108,119function ................................................................................98–114microarrays ................................................................................122profiling ............................................................................... 115–131qRT-PCR ..................................................................................123

miRNAinhibitorlibrary............................................................................110miRRNAicassettes................................................................................ 62mirVana™miRNAIsolationKit........................................................................112MultiSiteGateway®Three-FragmentVectorConstructionKit..................................................... 64

NNCode™EXPRESSSYBR®GreenER™miRNAqRT-PCRKit........................................................123NCode™miRNAAmplificationSystem............................................................... 118–119NCode™miRNAmicroarrays...................................................................... 120–122NCode™ncRNAarrayanalysis ..................................................................... 124–130NCode™platform ............................................................................115,120NCode™Profilersoftware...........................................................................122NCode™RapidmiRNALabelingSystem.............................................................. 118–119NCode™VILO™miRNAcDNASynthesisKit ................................................................123Neon™TransfectionSystem......................................................................10,76–78next-generationsequencing........................................................................126non-codingRNAs..........................................................120,123–125,150. See also miRNAnucleicacidpurification............................................................................ 91NuPAGE®Novex®gels .............................................................................. 91

Ooff-targeteffects............................................. 4,7–8,11,15,20,29–31,34,40,42–45,64–65,73,80,82,93Oligofectamine™TransfectionReagent ............................................................... 7,72,76oncoretrovirus .................................................................................. 17

PPCR...................................................................................... 18,116phosphorylation................................................................................151PlateSelect™RNAi................................................................................ 48Platinum®TaqDNAPolymerase....................................................................123,145PLUS™Reagent .................................................................................. 75pMIR-REPORT™miRNAExpressionReporterVectorSystem.....................................................110PolIImiRRNAiexpressionvectors. See BLOCK-iT™PolIImiRRNAiexpressionvectorsPolIImiRRNAivectors.........................................................................85,94–95pre-miRNAs............................................................................... 56,98–114Pre-miR™miRNAPrecursors......................................................................109,110proteinseparation................................................................................ 91pSCREEN-iT™/lacZ-DESTGateway®VectorKit............................................................... 55PureLink™reagentsandkits................................................................... 142–143,146

QqPCR ..........................................................21,24,120,123–124,126,130,135,152–153,155qRT-PCR.........................................9,29–30,34–36,41,68,73,79,88–91,93,96,114,115–116,123–125,130Quant-iT™RNAAssayKit............................................................................ 24

159

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Rreal-timePCR/RT-PCR................................................ 18,47,68,82,89–90,104–106,108,111,117,113RecoverAll™TotalNucleicAcidIsolationKit...............................................................113retrovirus ...................................................................................... 22RISC. See RNA-inducedsilencingcomplexRNAi .................................................................... 40–52,53–71,72–78,79–87,88–91

cassette............................................................................... 18,55,60controls................................................................................. 79–87delivery...............................................................................68,72–78functionalvalidation........................................................................... 96measuringknockdown....................................................................... 88–91services................................................................................. 92–96transfection................................................................................. 84vectors...........................................................................68,70,72–78,85

RNAiDesigner. See BLOCK-iT™RNAiDesignerRNAi,in vivo. See also DNAmethylation,chromatinremodeling,noncodingRNAsRNA-inducedsilencingcomplex(RISC)......................................................... 2,6,19,56,98,109RNAinterference. See RNAiRNAi™siRNA,BLOCK-iT™siRNA........................................................................ 92RNAPolymeraseII........................................................................... 56,98–114RT-PCR...........................................................................82,108,111,113,117

SSangermiRBaseSequenceDatabase................................................................ 120–121sequencing.........................................................53,102,120,126,136,138–142,144–146,152

bisulfite..................................................................................146services

assaydevelopment ............................................................................ 97BLOCK-iT™RNAidesign......................................................................... 48BLOCK-iT™RNAiDesigner ........................................................................ 66customRNAi................................................................................ 55DNAmethylation.........................................................................134,136miRNA .................................................22,40,43,53,56,60–62,64–65,75,80,102,110,111,116miRNAControlVectors.......................................................................... 85miRNAi ................................................................................... 27RNAi..................................................................................... 85RNAidesign..........................................................................8,40,45,93RNAifunctionalvalidation ....................................................................... 96RNAisubcloning ............................................................................. 94RNAitransfection...........................................................................10,75RNAivectorcloningkits ..........................................................................8stablecelllinegeneration........................................................................ 97viralproduction............................................................................92,95

shRNA................................................ 3,6,7–8,18,19,22,27,30–34,55–56,62–63,66–71,72,75,93–97shRNAexpression.........................................................................18,68–69,71shRNAvectors. See BLOCK-iT™RNAivectors;See BLOCK-iT™shRNAvectorsSilencer®Pre-designedsiRNA.....................................................................48–49,52Silencer®Select.................................................................................. 82Silencer®SelectNegativeControlsiRNA................................................................... 79Silencer®SelectPositiveControlsiRNA.................................................................... 79Silencer®SelectPre-designedsiRNA.................................................................48–49,52Silencer®SelectsiRNA......................................................... 4,8–9,10–13,29–30,40–44,46,49Silencer®SelectsiRNAlibraries....................................................................... 50–51Silencer®SelectValidatedsiRNA ...................................................................... 48–49Silencer®siRNA................................................................................52,86Silencer®ValidatedsiRNA.......................................................................... 48–49

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Index

160


siRNA.............................. 2–6,7–9,10–16,19,20,23,25,27–28,29–37,40–52,72–76,80–82,85–87,92–93,96,109,114pools ...................................................................................51,75

siRNAin vivodelivery.............................................................................. 26siRNAlibraries ............................................................................29–32,48,50siRNAreportercontrols ............................................................................. 84SOLiD™RNAAnalysisSolution.................................................................... 101–102SOLiD™System ........................................................................100–103,136–141StealthRNAi™siRNA ..........................................8–9,10–13,19–20,22–23,26–28,40,44–49,73,75,83–85,93StealthRNAi™siRNAcontrols.......................................................................79,84StealthSelectRNAi™siRNA ...........................................................................4stressresponsepathways ....................................................................... 9,40,93sumoylation ...................................................................................150SuperScript®One-CyclecDNAKit...................................................................... 91SuperScript®PlusDirect ............................................................................ 91SuperScript®RNAAmplificationSystems .................................................................. 91SuperScript®VILO™Kit ............................................................................. 24SYBR®Green–basedPCRmastermixes................................................................... 90syntheticRNAi .............................................................................. 8,22,40

TTaqDNAPolymerase.............................................................................123TaqMan®ArrayMicroRNACards ......................................................................106TaqMan®Assay.................................................................................103TaqMan®GeneExpressionAssays.............................................................. 24,88–91,111TaqMan®GeneExpressionCells-to-Ct™Kit ................................................................113TaqMan®MGBProbes .............................................................................. 90TaqMan®MicroRNAArrays .......................................................................100,104TaqMan®MicroRNAAssays.................................................................... 102,106–107TaqMan®MicroRNACells-to-Ct™Kit....................................................................113TaqMan®probes............................................................................... 88–90TOPO®technology ...............................................................................146traditionalsiRNA................................................................................. 49transfection............................................ 3,5,7–8,10–16,18,22,30–34,52,68,79–87,93–94,97,109–110

conditions .......................................................7,14,32,36,67,72–73,75–76,79,84–85,94controls....................................................................................7efficiency..................................................3,7,10,11–12,15,16,65,72,74,76–77,80,82,85–86reagents......................................................3,7–8,10–12,11,22,33,73,75,79–81,84–85,94

TRIzol®PlusReagent............................................................................... 24TRIzol®Reagent..............................................................................116,128

Uubiquitination..................................................................................151

VValidatedStealthRNAi™siRNA........................................................................ 48viraldelivery ............................................................. 10–16,17–18,22,57,68–69,72,93–95ViraPower™T-REx™LentiviralExpressionSystem............................................................. 64

Wwesternblotting...................................................................... 70,88–91,111,154

XX-chromosomegenesilencing.......................................................................134

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