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Agilent Low RNAInput FluorescentLinear AmplificationKit

ProtocolProduct Number 5184-3523 Optimized for use with:Agilent oligonucleotide & cDNA microarrays

Version 2.0 Poly A+ RNA recommendations. Plant RNA quality recommendations.

August 2003

See list of components or package insert for storageconditions.

Research Use Only

2 Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol

Notices

© 2003 Agilent Technologies, Inc.

Manual Part Number5185-5818

EditionFirst Edition, Version 2.0, August 2003

Printed in USA

Agilent Technologies, Inc.Bioresearch Solutions Unit3500 Deer Creek Road, MS 25U7Palo Alto, CA 94304

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3Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol

Introduction 4

Kit Contents 5

Required Equipment 5

Safety Notes 6

General Procedural Notes 6

Fluorescent cRNA Synthesis 7

Other Required Reagents—Not Included In Kit 9

RNA Preparation and Qualification 9

cDNA Synthesis From Total RNA 10

Fluorescent cRNA Synthesis: in vitro transcription and incorporation of cyanine 3- or cyanine 5-CTP 11

Purification of Amplified cRNA 12

Quantifying cRNA Products 13

cRNA Amplification & Fluorescent cDNA Synthesis 14

Other Required Reagents—Not Included In Kit 16

Reagent Preparation 16Cyanine Dye Mix 16

Cyanine 3-dCTP 16Cyanine 5-dCTP 17

RNA Preparation and Qualification 17

cDNA Synthesis From Total RNA 18

cRNA Amplification 19

Purification of Amplified cRNA 19

Fluorescent cDNA Synthesis From Amplified cRNA 20

Purifying Labeled cDNA 21

Concentrating Cyanine 3- or Cyanine 5-labeled cDNA 22

Appendix I: RNA Quality 23

Appendix 2: Plant RNA Quality 24

Contact Information back page

Table of Contents


1Introduction

Agilent’s Low RNA Input Fluorescent Linear Amplification kit (5184-3523) generates fluorescentcRNA (complimentary RNA) for use with Agilent’s oligonucleotide microarrays or fluorescentcDNA for use with all of Agilent’s cDNA microarrays. This kit is to be used when the amount ofsample RNA ranges between 50 ng and 5 µg of total RNA, or a minimum of 10 ng of poly A+ RNA.

Two labeling procedures are covered in this protocol:1) Fluorescent cRNA Synthesis (Page 7) for use with Agilent Oligo Microarrays except

Arabidopsis 1 Oligo Microarrays.2) cRNA Amplification and Fluorescent cDNA Synthesis (Page 14) for use with Agilent

cDNA Microarrays.

A feature extraction software package links a feature to a design file and determines the relativefluorescence intensity of the two different dyes after normalization. This process allows you toquantitate differential gene expression between the two samples when compared.

An experienced user should require approximately 6-10 hours to complete this procedure fromstart to finish, depending on the number of samples being processed and the procedure beingperformed. If necessary, the procedure may be broken down into two steps with a break overnightat step 15 when following the fluorescent cRNA synthesis protocol or step 14 when using thecRNA amplification and fluorescent cDNA synthesis protocol.

There is routinely at least a 100-fold RNA amplification with use of this kit. Your results may varywith the purity of the RNA preparation that you use.

Reading the entire protocol before you commence work is essential for success.


CAUTION The components marked in red are used only when generatingfluorescently-labeled cDNA.

Make sure you use the correct dNTP for the first RT reaction.

Required Equipment• Ice and ice bucket• Powderless gloves• UV-Vis scanning spectrophotometer and 0.1 mL volume quartz microcuvettes (1 cm path

length) or the NanoDrop ND-1000 Spectrophotometer. We recommend using theNanoDrop Spectrophotometer because the amount of RNA used in the measurement isvery low. Traditional UV-Vis spectorphotometer cuvettes consume too much sampleunless a microcuvette can be used.

• Timer or clock• Black waterproof marking pen• Micropipettors to pipette a range of 0.1 µL to 1 mL volumes • Sterile, nuclease-free 1.5 mL microcentrifuge tubes• Sterile, nuclease-free aerosol barrier pipet tips• Heating block, temperature set to 65°C• Microcentrifuge• Circulating water baths, temperature set to 40°C and 65°C• Vortex mixer• SpeedVac rotary dessicator (needed if converting amplified cRNA to labeled cDNA)• (Optional) Refrigerated Microcentrifuge for 4ºC spin-downs during the

purification procedures.

Kit Contents

Agilent Low RNA Input Fluorescent Linear Amplification Kit (5184-3523)

Component Volume (µL)T7 Promoter Primer 100

5X First Strand Buffer 180

0.1 M DTT 210

10 mM dNTP mix 20

MMLV RT 20 (There are 2 vials)

RNaseOUT 20

4X Transcription Buffer 400

NTP mix 160

Inorganic Pyrophosphatase 12

T7 RNA Polymerase 16

50% PEG (polyethylene glycol) 128

CTP 12

Random Hexamers 20

dNTP 20

RNAse I “A” 20

Store all components at -20ºC. NOTE


Safety Notes1. Wear appropriate protective equipment when working in a laboratory.2. Cyanine 3-CTP, -dCTP and cyanine 5-CTP, -dCTP are possible carcinogens. Avoid inhala-

tion, swallowing or contact with skin.3. Dithiothreitol (DTT) causes irritation to the skin, eyes, and respiratory tract and is

harmful if swallowed or inhaled. It may also be harmful if absorbed through the skin,and may affect the central nervous system.

General Procedural Notes• Follow Biosafety Level 1 (BL1) safety rules.• We recommend preparing amplified RNA in batches of no less than 6.

This policy minimizes errors associated with pipetting small volumes of enzyme solutions. • Our procedure specifies reagent volumes for 1 reaction and 6.5 reactions. We have added

an extra half-reaction to the recipe to ensure that you do not run out of reagents in yourfinal master mix.

• To specify reagent volumes for “n” reactions, multiply the volumes for 1 reaction by (n + 0.5).

• To prevent contamination of reagents by ribonucleases, always wear powderlesslaboratory gloves. Use dedicated, nuclease-free solutions and pipettors with nuclease-freeaerosol-resistant tips.

• Maintain a clean work area. • Cyanine 3 and cyanine 5 are photolabile: minimize exposure to light. • Stock solutions that are stored frozen in 1.5 mL microcentrifuge tubes should be prepared

for use as follows:• Thaw the aliquot as rapidly as possible without heating it above room

temperature.• Vortex briefly. Microcentrifuge for 5 – 10 seconds to drive tube contents off the

tube wall and lid.• Store on ice until use.


For use with Agilent oligo microarrays (excluding Arabidopsis 1 oligo microarrays).

The Agilent Low RNA Input Fluorescent Amplification Kit Protocol has been written to includetwo unique labeling procedures—one to create fluorescently-labeled cRNA and another tocreate fluorescently-labeled cDNA (sense)—all in one convenient kit format.

The fluorescent cRNA synthesis procedure has been optimized to work with Agilent’s oligomicroarrays (except Agilent Arabidiposis 1 oligo microarrays). It features an efficient, singletube, single amplification, two-color labeling method to create ample cyanine 3- and cyanine 5-labeled cRNA. In this procedure, one sample is labeled with cyanine 3 (which is excited by a 532nm laser) and one with cyanine 5 (which is excited by a 633 nm laser). A primer, which containspoly dT and a T7 polymerase promoter, is annealed to the poly A+ RNA. Reverse transcriptase isadded to the reaction to synthesize the first and second strands of cDNA. At this point, double-stranded cDNA has been synthesized. Next, cRNA is synthesized using T7 RNA polymerase,which simultaneously incorporates cyanine 3- or cyanine 5-labeled CTP. Once labeling iscomplete, both samples are combined and hybridized to the microarray. Genes whoseexpresssion differs between the samples are easily identifiable by scanning the microarray witha laser-based detection system.

Please see the following page for a schematic drawing depicting the entire amplified cRNAProcedure.

READING THE ENTIRE PROTOCOL BEFORE YOU BEGIN IS ESSENTIAL TO SUCCESS

Workflow Time Table for Preparation of Fluorescent cRNA

Step Temperature Time

cDNA Synthesis (Total time: 155 minutes)Denature Primer and Template 65ºC 10 min.Snap Cool ice 5 min.Double-stranded cDNA Synthesis 40ºC 120 min.MMLV-RT Inactivation 65ºC 15 min.Snap Cool ice 5 min.

cRNA Synthesis (Total time: 120 minutes)cRNA Synthesis 40ºC 120 min.

cRNA Purification (Total time: 30 minutes)cRNA Purification RT 30 min.

Fluorescent cRNA Synthesis Procedure


Before getting startedThe following reagents are used for generating fluorescent labeled cDNA from cRNA: dNTPs,one tube of MMLV-RT, Random Primers, RNase 1 “A”. If you are generating labeled cRNA, pleaseplace these reagents in a separate box to minimize inadvertently using the wrong reagent. Storeat -20ºC.

Fluorescent cRNA is for use on Agilent oligo microarrays except Arabidopsis 1 oligo microarrays.

Other Required Reagents—Not included in Kit• Cyanine 3-CTP (10.0 mM), PerkinElmer/NEN Life Sciences

catalog number NEL580*, **• Cyanine 5-CTP (10.0 mM), PerkinElmer/NEN Life Sciences

catalog number NEL581*, *** Quote promotional number PerkinElmer/NEN AG2001 to receive a 30% discount

on these products. (European customers, please ask for Agilent’s preferred pricingwhen ordering these products, as the promotional codes are not recognized inEurope.)

** PerkinElmer/NEN provides sufficient cyanine 3-CTP & cyanine 5-CTP for FOUR (4)reactions; if you wish to do more than four cyanine 3-CTP & cyanine 5-CTPreactions, be sure to purchase more than one tube.

• Total RNA. We recommend a concentration between 5 ng/µL and 5000 ng/µL• Poly A+ RNA. We recommend a concentration between 2 ng/µL and 200 ng/µL• DNase/RNase-free distilled water, Invitrogen catalog number 10977015• Qiagen RNeasy mini kit, Qiagen catalog number 74104 or 74106• 100% Ethanol, Amresco, catalog number E193

RNA Preparation and QualificationIt is essential that your total or poly-A+ RNA be of high quality that meets these specifications:

1. Size distribution (see Appendix 1 & 2 for illustrations): Analyze using a denaturing gel or Agilent's 2100 bioanalyzer.

For routine RNA QC analysis, we recommend Agilent's 2100 bioanalyzer with an RNA 6000Nano LabChip® Kit (product number 5065-4776). The bioanalyzer provides a complete RNAprofile with as little as 5 ng/µL total or poly A+ RNA and can quickly reveal sample degradation. Where sample is limited, we recommend the RNA 6000 Pico LabChip Kit (product number 5065-4473) for qualitative measurement of RNA (200-5000 pg/µL total RNA) (500-5000 pg/µL mRNA).

• For total RNA, ribosomal RNA should be visible at approximately 1.9 and 5 kb. The bandsshould be sharp and clear. If they are smeared or there are multiple peaks, then the RNAhas been degraded. In addition, high molecular weight bands (>9000 kb) indicate DNAcontamination in the sample.

• For poly A+ RNA, a faint smear in the range of 0.5 to 2 kb should be detectable.

Necessary Plant RNA Purification Steps: It is essential that you follow the recommendedRNA clean-up steps in Appendix 2 when using plant RNA. By failing to follow these RNAclean-up steps, it is likely that impure RNA will be introduced into your labeling reaction,thus affecting downstream microarray results.

2 . Determine the RNA concentration:

Measure UV absorbance at 260 nm using a spectrophotometer. An A260 of 1 equals an RNAconcentration of approximately 40 µg/mL.

Plant RNA: Consult Appendix 2 for guidance.

NOTE

NOTE


CAUTION


cDNA Synthesis From Total RNA(Time required: ~3 hours)

1. To a 1.5 mL microcentrifuge tube, add 50 to 500 ng total RNA in a volume of 10.3 µL orless. The total concentration should be at least 5 ng/µL.See the Introduction for details.

If you want to use more than 500 ng total RNA or 10 ng or more or poly A+ RNA, the totalvolume should be 6.5 µL.

2. Add 1.2 µL of T7 Promoter Primer (from kit).

If the total RNA input is greater than 500 ng (5 µg is the maximum) or if the poly A+ RNA input isat least 10 ng, add 5 µL of T7 Promoter Primer.

3. Use nuclease-free water to bring the total reaction volume to 11.5 µL.

4. Denature the primer and the template by incubating the reaction at 65°C in a heatingblock for 10 minutes.

5. Place the reactions on ice and incubate for 5 minutes.

6. Immediately—prior to use, gently mix the following components by pipetting, in theorder indicated, at room temperature:

Pre-warm the 5X First Strand Buffer by incubating the vial in a 65°C waterbath for 3-4 minutes.To ensure optimal re-suspension, vortex briefly and spin the tube briefly in a microfuge to drivethe contents off the tube walls. Keep at room temperature until use.

There are two vials of dNTPs. Be sure to use the 10mM dNTP mix.

7. To each sample tube, add 8.5 µL cDNA Master Mix.

8. Incubate samples at 40°C in a circulating water bath for 2 hours.

9. Move samples to a heating block or water bath set to 65°C and incubate for 15 minutes.

Incubation at 65°C inactivates MMLV-RT.

10. Move samples to ice. Incubate on ice for 5 minutes.

11. Spin samples briefly in a microcentrifuge to drive tube contents off the tube wall and lid.

NOTE

NOTE

NOTE

NOTE

cDNA Master Mix

Component Vol. (µL/rxn) Vol. (µL/6.5 rxn)

5X First Strand Buffer 4.0 26

0.1 M DTT 2.0 13

10 mM dNTP mix 1.0 6.5

MMLV RT 1.0 6.5

RNaseOUT 0.5 3.3

TOTAL VOLUME 8.5 55.3


NOTE


Fluorescent cRNA Synthesis: in vitro transcription and incorporationof cyanine 3- or cyanine 5-CTP(Time required: 2.5 hours)

12. To each sample tube, add EITHER 2.4 µL cyanine 3-CTP (10 mM) OR 2.4 µL cyanine 5-CTP (10 mM).

Cyanine 3 is bright pink and cyanine 5 is bright blue. Both are light sensitive. Minimize lightexposure.

Pre-warm the 50% PEG solution by incubating the vial in a 40°C waterbath for 1 minute. Toensure optimal re-suspension, vortex briefly and spin the tube briefly in a microfuge to drive thecontents off the tube walls. Keep at room temperature until use.


Do not add enzymes until just before you do the reaction.

14. To each sample tube, add 57.6 µL of Transcription Master Mix. Gently mix by pipetting.

15. Incubate samples in a circulating water bath at 40°C for 2 hours.

Protect the samples from light by covering the water bath.

Transcription Master Mix

Component Vol. (µL/rxn) Vol. (µL/6.5 rxn)Nuclease-free water 15.3 99.44X Transcription Buffer 20 1300.1 M DTT 6.0 39NTP Mix 8.0 5250% PEG 6.4 41.6RNAseOUT 0.5 3.3Inorganic Pyrophosphatase 0.6 3.9T7 RNA Polymerase 0.8 5.2TOTAL VOLUME 57.6 374.4

NOTE

NOTE

NOTE


NOTE


Purification of Amplified cRNA(Time required: ~ 30 minutes)

We recommend using Qiagen’s RNeasy mini spin columns for purifying labeled cRNA samples.Unincorporated dye-labeled nucleotides in the hybridization solution significantly increasebackground fluorescence on the microarray. Please follow the RNeasy mini kit protocol(reproduced here) with our modifications.

We have observed higher yields when using a refigerated centrifugation procedure, Therefore,we highly recommend using a refrigerated microcentrifuge set at 4ºC for the centrifugation stepslisted below. Alternatively, you could locate your microcentrifuge in a 4ºC cold room toaccomodate this recommended procedure.

Remember to add four volumes of ethanol to Buffer RPE before using the kit for the first time(See bottle label for specific volume).

16. Add 20 µL of nuclease free-water to your cRNA sample, to obtain a total volume of 100 µL.

17. Add 350 µL of Buffer RLT and mix throughly.

18. Add 250 µL of ethanol (96-100% purity) and mix thoroughly by pipetting. DO NOTCentrifuge.

19. Transfer 700 µL of cRNA sample to an RNeasy mini column in a 2 mL collection tube.Centrifuge the sample for 30 seconds at 13,000 rpm. Discard the flow-through andcollection tube.

20. Transfer the RNeasy column to a new collection tube and add 500 µL of buffer RPE tothe column. Centrifuge the sample for 30 seconds at 13,000 rpm. Discard the flowthrough. Re-use the collection tube.

21. Again, add 500 µL of Buffer RPE to the column. Centrifuge the sample for 60 seconds at13,000 rpm. Discard the flow through and the collection tube.

22. Elute the cleaned cRNA sample by transferring the RNeasy column to a new 1.5 mLcollection tube. Add 30 µL RNase-free water directly onto the RNeasy filter membrane.Wait 60 seconds before centrifuging for 30 seconds at 13,000 rpm. SAVE THE FLOW-THROUGH and the collection tube.

23. Again, add 30 µL RNase-free water directly onto the RNeasy filter membrane. Wait 60seconds before centrifuging for 30 seconds at 13,000 rpm. The total final flow throughvolume should be approximately 60 µL. Store at -80°C until needed. Discard the RNeasycolumn.

NOTE


NOTE


Quantitating cRNA ProductsA typical cRNA amplification reaction, starting with 50 ng of RNA, with Agilent’s kit will yield 0.2 - 1 µg of cRNA depending on the purity and quality of the input sample RNA. This amount ofcRNA is typically too low to be quantitated using a spectrophotometer with a standard cuvette.We recommend quantitating cRNA using a NanoDrop Spectrophotometer (to minimize theamount of sample consumed by the measurement).

24. Use 1 µL nuclease free water to blank the NanoDrop instrument. Then use 1 µL ofamplified cRNA for analysis. Calculate the concentration of cRNA by using the formula

1 OD260 = (10) x 40 µg/mL RNA.

• The reaction is multiplied by 10 because the pathlength of analysis is 1 mm.

Excessive freeze-thaw cycles may reduce cRNA integrity. If you are unsure about your product,we suggest re-evaluation on an Agilent 2100 bioanalyzer.



For use with Agilent cDNA Microarrays

The Agilent Low RNA Input Fluorescent Amplification Kit Protocol has been written to includetwo unique labeling procedures—one to create fluorescently-labeled cRNA and another tocreate fluorescently-labeled cDNA (sense)—all in one convenient kit format.

The cRNA Amplification & Fluorescent cDNA Synthesis Procedure has been optimized to workwith Agilent’s cDNA microarrays. It features an efficient, single tube, single amplification, two-color labeling method to create ample cyanine 3- and cyanine 5-labeled cDNA (sense). In thisprocedure, one sample is labeled with cyanine 3 (which is excited by a 532 nm laser) and onewith cyanine 5 (which is excited by a 633 nm laser). A primer, which contains poly dT and a T7polymerase promoter, is annealed to the poly A+ RNA. Reverse transcriptase is added to thereaction to synthesize the first and second strands of cDNA. At this point, double-strandedcDNA has been synthesized. Next, cRNA is synthesized in the absence of labeled nucleotides, togenerate unlabeled cRNA. Then, reverse transcriptase, random primers and cyanine 3-dCTP orcyanine 5-dCTP are added to the reaction and the cRNA is converted to labeled cDNA (sensestrand). Once labeling is complete, both samples are combined and hybridized to the microarray.Genes whose expression differs between the samples are easily identifiable by scanning themicroarray with a laser-based detection system.

Please see the following page for a schematic drawing depicting the entire amplified cDNAProcedure.

READING THE ENTIRE PROTOCOL BEFORE YOU BEGIN IS ESSENTIAL TO SUCCESS

Workflow Time Table for Preparation of Fluorescent cDNA

Step Temperature Time

cDNA Synthesis (Total time: 155 minutes)Denature Primer and Template 65ºC 10 min.Snap Cool Ice 5 min.

Double-stranded cDNASynthesis

40ºC 120 min.

MMLV-RT Inactivation 65ºC 15 min.Snap Cool Ice 5 min.

cRNA Synthesis (Total time: 120 minutes)cRNA Synthesis 40ºC 120 min.

cRNA Purification (Total time: 30 minutes)cRNA Purification RT 30 min.

DNA Synthesis (Total time: 125 minutes)Denature Primer & Template 65ºC 10 min.Snap Cool Ice 5 min.DNA Synthesis 40ºC 60 min.MMLV-RT Inactivation 65ºC 15 min.Snap Cool Ice 5 min.RNA Digestion RT 30 min.

DNA Purification (Total time: 90 minutes)DNA Purification RT 30 min.SpeedVac DNA 45ºC 60 min.

cRNA Amplification & Fluorescent cDNA Synthesis


Before getting startedFluorescent cDNA is for use with Agilent cDNA microarrays only.

Other Required Reagents—Not included in Kit• TE Buffer (10 mM Tris; 1 mM EDTA; pH 8.0, nuclease-free, Amresco

catalog number E112• Cyanine-3-dCTP (1.0 mM), Perkin Elmer/NEN Life Sciences

catalog number NEL576*• Cyanine-5-dCTP (1.0 mM), PerkinElmer/NEN Life Science

catalog number NEL577** Quote promotional number PerkinElmer/NEN AG2001 to receive a 30% discount

on these products. (European customers, please ask for Agilent’s preferred pricingwhen ordering these products, as the promotional codes are not recognized inEurope.)

• Total RNA. We recommend a concentration between 5 ng/µL and 5000 ng/µL• Poly A+ RNA. We recommend a concentration between 2 ng/µL and 200 ng/µL• DNase/RNase-free distilled water, Invitrogen catalog number 10977015• Qiagen RNeasy mini kit, Qiagen catalog number 74104 or 74106• Qiagen QiaQuick PCR kit, Qiagen catalog number 28104• 100% Ethanol, Amresco catalog number E193

Reagent PreparationNotes:

• These reagents may be prepared during the 2 hour incubation period in step 8 (cDNAsynthesis).

• Diluted cyanine 3-dCTP and cyanine 5-dCTP solutions may be prepared ahead of time andstored at -80°C.

• You will need to add 1.25 µL of diluted cyanine 3- or cyanine 5-dCTP to each sample. Besure to dilute enough dye-labeled dCTP to accommodate your reactions.

Cyanine Dye Mix(Time required: 5 – 10 minutes)Do this step only if you plan to generate fluorescently-labeled cDNA from amplified cRNA

Cyanine 3-dCTP, 500 µM(Enough for 20 labeling reactions)

1. Thaw 1 mM cyanine 3-dCTP stock and place on ice.

2. Vortex briefly.

3. Spin briefly (1 – 2 seconds) at maximum speed in a microcentrifuge to drive contents offthe tube wall and lid.

4. Pipet 12.5 µL cyanine 3-dCTP stock solution into a microcentrifuge tube.

5. Add 12.5 µL nuclease-free water and mix gently.

6. Store frozen in the dark at -80º C. Consult your cyanine dye supplier for long termstorage and stability conditions.



Cyanine 5-dCTP, 500 µM(Enough for 20 labeling reactions)

1. Thaw 1 mM cyanine 5-dCTP stock and place on ice.

2. Vortex briefly.

3. Spin briefly (1 – 2 seconds) at maximum speed in a microcentrifuge to spin downcontents.

4. Pipet 12.5 µL cyanine 5-dCTP stock solution into a microcentrifuge tube.

5. Add 12.5 µL nuclease-free water and mix gently.

6. Store frozen in the dark at -80º C. Consult your cyanine dye supplier for long termstorage and stability conditions.

RNA Preparation and QualificationIt is essential that your total or poly-A+ RNA be of high quality that meets these specifications:

1. Size distribution (see Appendix 1 & 2 for illustrations): Analyze using a denaturing gel or Agilent's 2100 bioanalyzer.

For routine RNA QC analysis, we recommend Agilent's 2100 bioanalyzer with an RNA 6000Nano LabChip® Kit (product number 5065-4776). The bioanalyzer provides a complete RNAprofile with as little as 5 ng/µL total or poly A+ RNA and can quickly reveal sample degrada-tion. Where sample is limited, we recommend the RNA 6000 Pico LabChip Kit (product num-ber 5065-4473) for qualitative measurement of RNA (200-5000 pg/µL total RNA) (500-5000pg/µL mRNA).

• For total RNA, ribosomal RNA should be visible at approximately 1.9 and 5 kb. The bandsshould be sharp and clear. If they are smeared or there are multiple peaks, then the RNAhas been degraded. In addition, high molecular weight bands (>9000 kb) indicate DNAcontamination in the sample.

• For poly A+ RNA, a faint smear in the range of 0.5 to 2 kb should be detectable.

Necessary Plant RNA Purification Steps: It is essential that you follow the recommendedRNA clean-up steps in Appendix 2 when using plant RNA. By failing to follow these RNAclean-up steps, it is likely that impure RNA will be introduced into your labeling reaction,thus affecting downstream microarray results.

2 . Determine the RNA concentration:

Measure UV absorbance at 260 nm using a spectrophotometer. An A260 of 1 equals an RNAconcentration of approximately 40 µg/mL.

Plant RNA: Consult Appendix 2 for guidance.


CAUTION


cDNA Synthesis From Total RNA(Time required: ~3 hours)

1. To a 1.5 mL microcentrifuge tube, add 50 to 500 ng total RNA in a volume of 10.3 µL orless. The total concentration should be at least 5 ng/µL.See the Introduction for details.

If you want to use more than 500 ng total RNA or 10 ng or more of poly A+ RNA, the totalvolume should be 6.5 µL.

2. Add 1.2 µL of T7 Promoter Primer (from kit).

If the total RNA input is greater than 500 ng (5 µg is the maximum) or if the poly A+ RNA input isat least 10 ng, add 5 µL of T7 Promoter Primer.

3. Use nuclease-free water to bring the total reaction volume to 11.5 µL.

4. Denature the primer and the template by incubating the reaction at 65°C in a heatingblock for 10 minutes.

5. Place the reactions on ice and incubate for 5 minutes.


Pre-warm the 5X First Strand Buffer by incubating the vial in a 65°C waterbath for 3-4 minutes.To ensure optimal re-suspension, vortex briefly and spin the tube briefly in a microfuge to drivethe contents off the tube walls. Keep at room temperature until use.

There are two vials of dNTPs. Be sure to use the 10mM dNTP mix.

7. To each sample tube, add 8.5 µL cDNA Master Mix.

8. Incubate samples at 40°C in a circulating water bath for 2 hours.

9. Move samples to 65°C. Incubate at 65°C for 15 minutes.

Incubation at 65°C inactivates MMLV-RT.

10. Move samples to ice. Incubate on ice for 5 minutes.

11. Spin samples briefly in a microcentrifuge to drive tube contents off the tube wall and lid.

cDNA Master Mix

Component Vol. (µL/rxn) Vol. (µL/6.5 rxn)5X First Strand Buffer 4.0 260.1 M DTT 2.0 1310 mM dNTP mix 1.0 6.5MMLV RT 1.0 6.5RNaseOUT 0.5 3.3TOTAL VOLUME 8.5 68.3

NOTE

NOTE

NOTE

NOTE

NOTE



cRNA Amplification(Time Required: 2.5 hours)Pre-warm the 50% PEG solution by incubating the vial in a 40°C waterbath for 1 minute. Toensure optimal re-suspension, vortex briefly and spin the tube briefly in a microfuge to drive thecontents off the tube walls. Keep at room temperature until use.


Do not add enzymes until just before you do the reaction.

13. To each sample tube, add 60 µL of Transcription Master Mix. Gently mix by pipetting.

14. Incubate samples in a circulating water bath at 40°C for 2 hours.

Purification of Amplified cRNA

We recommend using Qiagen’s RNeasy mini spin columns for purifying amplified cRNA samples.Unincorporated dye-labeled nucleotides in the hybridization solution significantly increasesbackground fluorescence on the microarray. Please follow the RNeasy mini kit protocol(reproduced here) with our modifications.

We have observed higher yields when using a refigerated centrifugation procedure, Therefore,we highly recommend using a refrigerated microcentrifuge set at 4ºC for the centrifugation stepslisted below. Alternatively, you could locate your microcentrifuge in a 4ºC cold room toaccomodate this recommended procedure.

Transcription Master Mix

Component Vol. (µL/rxn) Vol. (µL/6.5 rxn)Nuclease-free water 12.1 78.64X Transcription Buffer 20 1300.1 M DTT 6.0 39NTP Mix 8.0 52CTP 5.6 36.450% PEG 6.4 41.6RNaseOUT 0.5 3.3Inorganic Pyrophosphatase 0.6 3.9T7 RNA Polymerase 0.8 5.2TOTAL VOLUME 60 390

NOTE

NOTE

NOTE



Remember to add four volumes of ethanol to Buffer RPE before using the kit for the first time(See bottle label for specific volume).

15. Add 20 µL of nuclease free-water to your cRNA sample, to obtain a total volume of100 µL.

16. Add 350 µL of Buffer RLT and mix well.

17. Add 250 µL of ethanol (96-100% purity) and mix thoroughly by pipetting. DO NOTCentrifuge

18. Transfer 700 µL of cRNA sample to an RNeasy mini column in a 2 mL collection tube.Centrifuge the sample for 30 seconds at 13,000 rpm. Discard the flow-through andcollection tube.

19. Transfer the RNeasy column to a new collection tube and add 500 µL of buffer RPE tothe column. Centrifuge the sample for 30 seconds at 13,000 rpm. Discard the flowthrough. Re-use the collection tube.

20. Again, add 500 µL of buffer RPE to the column. Centrifuge the sample for 60 seconds at13,000 rpm. Discard the flow through and the collection tube.

21. Elute the cleaned cRNA sample by transferring the RNeasy column to a new 1.5 mLcollection tube. Add 30 µL RNase-free water directly onto the RNeasy filter membrane.Wait 60 seconds before centrifuging for 30 seconds at 13,000 rpm. SAVE THE FLOW-THROUGH and the collection tube.

22. Again, add 30 µL RNase-free water directly onto the RNeasy filter membrane. Wait 60seconds before centrifuging for 30 seconds at 13,000 rpm. Discard the RNeasy column.

Fluorescent cDNA synthesis from amplified cRNA (Time Required: ~2.5 hours)The unlabeled cRNA concentration should be between 37.7 ng/µL and 500 ng/µL. If theconcentration is below 37.7 ng/µL, the amplified cRNA can be concentrated by drying thesolution under vacuum, at room temperature, in a rotary dessicator until dry (approximately 60minutes). Over-drying the cRNA can result in difficulty while re-suspending the cRNA. Re-suspend the cRNA in 10 µL of TE Buffer.

If you have less than 500 ng of amplified cRNA, use the entire reaction in this labeling step.

1. To each sterile nuclease-free reaction tube, add 500 ng of cRNA and bring the totalsample volume to 13.25 µL with nuclease-free water.

2. Add 1.0 µL of Random Hexamers.

3. Incubate tube at 65°C for 10 minutes to denature the primer and the template RNA.

4. Place the reaction tubes on ice for 5 minutes.

5. Add 1.25 µL of either cyanine 3-dCTP (500 µM) or cyanine 5-dCTP (500 µM). Label eachtube appropriately.

NOTE

NOTE



Cyanine 3 is bright pink and cyanine 5 is bright blue. Both are light sensitive. Minimize lightexposure.

Pre-warm the 5X First Strand Buffer by incubating the vial in a 65°C waterbath for 3-4 minutes.To ensure optimal resuspension vortex briefly and spin the tube briefly in a microfuge to drivethe contents off the tube walls. Keep at room temperature until use.

6. Immediately prior to use, gently mix the following components by pipetting, in the orderindicated, at room temperature:

There are two vials of dNTPs. Be sure to use the vial marked "dNTP.” DO NOT USE the 10mMdNTP mix.

7. Aliquot 9.5 µL of cDNA Master Mix into each sample tube. Incubate cDNA synthesis reaction at 40°C in a waterbath for 60 minutes.

8. Move the reaction tubes to a waterbath or a heating block set to 65°C. Incubate for 10minutes.

9. Place the reaction tubes on ice for 5 minutes.

10. Add 1.0 µL RNase I “A” to each reaction tube. Mix by pipetting. Incubate at roomtemperature for 30 minutes to degrade the RNA.

Purifying Labeled cDNA(Time Required: ~ 1.5 hours)We recommend using Qiagen’s QIAquick spin columns for purifying labeled cDNA samples.Unincorporated dye-labeled nucleotides in the hybridization solution significantly increasebackground fluorescence on the microarray. Please follow the QIAquick PCR Purification KitProtocol (reproduced here) with our modifications.

Remember to add ethanol to Buffer PE before use. (See bottle label for volume.)

11. Combine the cyanine 3- and cyanine 5-cDNA reactions for each microarray hybridization(50 µL).

12. Add 5 volumes of Buffer PB (250 µl) to 1 volume of the combined cyanine 3- andcyanine 5-cDNA labeling reactions (50 µL) and mix.

13. Place a QIAquick spin column in a 2 mL collection tube.

cDNA Master Mix

Component Vol. (µL/rxn) Vol. (µL/6.5 rxn)5X First Strand Buffer 5.0 32.50.1 M DTT 2.5 16.25dNTP 1.0 6.5MMLV RT 1.0 6.5TOTAL VOLUME 9.5 61.75

NOTE

NOTE

NOTE


NOTE


14. To bind the DNA, apply the sample to the QIAquick column and centrifuge it in amicrocentrifuge for 30 - 60 seconds at approximately 13,000 rpm (at least 10,000 x g).

15. Discard flow-through and place the QIAquick column back into the same collectiontube.

16. Wash the column with 0.40 mL of Qiagen’s Buffer PE and centrifuge for 60 seconds asdescribed in the protocol.

17. Discard flow-through, place the QIAquick column back into the same collection tube.Wash the column again with 0.40 mL of Qiagen’s Buffer PE and centrifuge for another60 seconds.

Be careful not to splash the flow-through back into the tip of the column. If flow-through doescome in contact with the tip, briefly spin the column and collection tube for 15 seconds prior totransfering to a new collection tube.

18. Transfer the QIAquick column from the used collection tube into a clean collection tube.

19. To elute the sample, add 30 µL of the Qiagen Buffer EB (10mM Tris-Cl, pH 8.5) to thecenter of the column, let sit for 1 minute, then centrifuge for 60 seconds.

20. Repeat step 19 with an additional 30 µL of Qiagen Buffer EB and elute into the sametube. The final eluted volume should be approximately 60 µL.

Concentrating Cyanine 3- or Cyanine 5-labeled cDNA(Time Required: ~ 1 hour)

21. Dry the solution under vacuum in a rotary dessicator until dry (approximately 60minutes).

To prevent cyanine dye degradation, do not use heat during drying.

22. Proceed directly to hybridization or freeze on dry ice and store at –80°C.

Due to the minute amount of sample material used in this protocol, you may not see a tintedpellet after sample concentration. However, the sample is present and you may continue withhybridization.

NOTE

NOTE

NOTE



Appendix 1:RNA Quality

Data from a high quality total RNA preparationAlthough a wide variety of ribosomal peak heights and ratios exist across a broad range of RNAsample types, most high quality RNA samples share the same common features.The most distinct and intense features are the 18S and 28S ribosomal peaks (16S & 23S, prokary-otic). The baseline between 29 seconds and the 18S ribosome is relatively flat and free of smallrounded peaks corresponding to smaller RNA molecules and there is an absence of smaller well-defined peaks between the two ribosomal peaks. Any RNA migrating between the ribosomalpeaks will be smooth and lack distinct peaks. Finally, depending on the RNA extraction method,the small 5S, 5.8S and tRNA may be present in the electropherogram from 24-27 seconds.

Data from a high quality mRNA preparationHigh quality mRNA run on the Agilent 2100 bioanalyzer typically has the shape of a broad peak,with transcripts falling in the range of 500 – 9000 bases long. The majority of transcript densityfalls in the size range of 1000 – 4000 bases. The electorpherograms of high quality samples aregenerally smooth and free of multiple large peaks. It is common for high quality mRNA samplesto contain low levels of ribosomal RNA contamination, which are characterized by the presenceof one to two large, well-defined ribosomal RNA peaks. The bioanalyzer software identifies andquantitates ribosomal peaks that are 5 percent of the total mRNA concentration or greater.

Mouse Spleen Total RNA

Bovine Brain mRNA


Appendix 2:Plant RNA Quality

Consistent with all RNA handling procedures, be careful when working with plant RNA to avoidintroducing nucleases that will degrade your sample. Before labeling, be sure to check thequality and integrity of your sample via gel electrophoresis or using the Agilent 2100 bioanalyzerand RNA 6000 Nano or RNA 6000 Pico kit (www.agilent.com/chem/chip). Pictured here areexamples of Arabidopsis RNA which may give you guidance when analyzing other plant RNAsamples. High quality Arabidopsis RNA will have two prominent ribosomal bands accompaniedby a series of smaller plastid ribosomal peaks that are isolated from the Arabidopsischloroplasts. Notice the difference in the baseline fluorescence of the degraded ArabidopsisRNA in comparison to the high quality RNA.

Typically, purified RNA from plant and fungal samples is contaminated with high concentrationsof cellulose, polysaccharides and interfering substances that are co-isolated with the RNA.Polysaccharides and secondary metabolites can interfere with the OD readings leading to errorsin concentration determination. Cellulose, polysaccharides and some other plant RNAcompounds will make the labeling reaction less efficient and should be removed prior tolabeling.

Degraded Arabidopsis RNA

High Quality Arabidopsis RNA


Plant RNA Isolation1. It is essential that you properly prepare your plant RNA for use in labeling reactions.

Available kits to isolate plant RNA include: Ambion, Inc. RNAqueous Kit, catalog number1912 used in conjunction with the Plant RNA Isolation Aid, catalog number 9690; Qiagen Inc.RNeasy Plant Mini Kit, catalog numbers 74903 & 74904.

2. After isolating the plant total RNA, perform an “initial RNA clean-up” step to eliminateexcess plant contaminants.

• We stongly recommend that you perform this extra clean-up step prior to isolating poly A+ RNA.

• If you are isolating poly A+ RNA directly from plant tissues, we strongly recommend that you perform this extra clean-up step with your isolated poly A+ RNA.

Available kits include: Qiagen Inc. RNeasy Kit, catalog numbers 74104 & 74106.

3. Determining the RNA Concentration: Measure the UV absorbance of the purified RNAsample at 230 nm, 260 nm, 280 nm, and 320 nm, either as single wavelength determinationsor as part of a wavelength scan (220 nm to 320 nm) using a UV/Visible spectrophotometer.An A260 of 1 equals an RNA concentration of approximately 40 µg/mL.

• The ratio between the readings at 260 nm and 280 nm provides an estimate of the purity of the nucleic acid. Pure preparations of RNA (and DNA) have OD ratio values (A260/A280) of between 1.8 and 2.0. Values below this indicate contamination of the sample with either phenol and/or protein. Values above this may indicate significant contamination of the sample with soluble carbohydrates.

• OD ratio values (A260/A230) less than 2.0 may indicate contamination by polysaccharides.

• Significant absorption caused by light scattering at 320 nm indicates the presence of particulate matter in the sample.

For reliable estimation, concentrations of at least 1 µg/mL are required.

Secondary RNA Clean-up: There are instances where certain tissue or plant RNA isolationmethods where the subsequent “initial clean-up” procedure may not be sufficient to remove allRNA contaminants. In these cases, we strongly recommend using two (2) Qiagen RNeasyclean-up columns to ensure that all contaminants have been removed for optimal RNA quality.

NOTE


Notes

Agilent TechnologiesBioresearch Solutions Unit3500 Deer Creek RoadPalo Alto, CA 94304E-mail: [email protected]

Agilent Gene Expression Microarrays Website:www.agilent.com/chem/dna

Information, descriptions and specifications aresubject to change without notice. Pleaseregister online with Agilent to receive newproduct updates at:www.agilent.com/chem/dnasupport

Part Number: 5185-5818 August 2003, Rev. 2.0

5185-5818

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