sure silencing
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Pathway-Centric Tools and Technology™
SureSilencing™ shRNATechnology Overview
Guaranteed Plasmid-Based RNA InterferenceFor EVERY Human, Mouse, and Rat Gene
Pathway-Centric Tools and Technology™
Topics to be Covered
Challenges Facing RNA Interference ResearchersSolutions SureSilencing shRNA ProvidesHow SureSilencing shRNA Works
Vector, Design Algorithm, Validation ProcessGuaranteed Success
How YOU Can Use SureSilencing shRNA
Pathway-Centric Tools and Technology™
RNA Interference ChallengesEffectiveness:Reconciling differences between knockdown efficiencies advertised by companies and observed by researchers.
Specificity:Insuring that the observed phenotype is due to the knockdown of only the gene of interest.Addressing “off-target” side-effects for publication purposes.
Applicability:Using RNA interference in a wider variety of cell lines with less than perfect transfection efficiencies.
Pathway-Centric Tools and Technology™
What SureSilencing shRNA ProvidesGuaranteed Performance:Suppress expression of gene of interest by at least 70 percent.
Two Successful Gene-Specific Designs:Ability to test if a different design provides same results.Control for non-specific and off-target effects.
Plasmid-Based System:Includes mammalian markers to select or enrich transfectants.Standard plasmid-based and lipid-mediated transfection.Renewable source of RNA Interference.
Pathway-Centric Tools and Technology™
THE SureSilencing shRNA GUARANTEE
At least two of the four provided SureSilencing™shRNA Plasmids are guaranteed to knock down the expression of the targeted gene at least 70 percent at the RNA level as measured by real-time qRT-PCR by in transfected cells upon FACS-based enrichment for GFP expression or selection for neomycin or puromycin resistance.
Pathway-Centric Tools and Technology™
What is SureSilencing™ shRNA?
Pre-designed shRNA constructs specific for a given target geneFour (4) pre-designed shRNA are provided on separate plasmids for every human, mouse, or rat gene.Every order also includes one negative control shRNA:A scrambled artificial sequence with no sequence identity to either one of the three genomesAll separately cloned into a mammalian expression vector system with the same selectable marker
Pathway-Centric Tools and Technology™
SureSilencing shRNA Plasmid BackbonesLife-time supply with single purchase
Bacterial origin of replication and ampicillin-resistance markerChoice of one of three mammalian markers
Neomycin for stable transfectionsPuromycin alternative marker for stable transfectionsGFP for transient transfections
Minimize non-specific off-target and toxic side effectsU1 promoter, transcribed by RNA Polymerase II, that normally transcribes mRNAProvides moderate shRNA expression levelOther promoter system express too much shRNA
Pathway-Centric Tools and Technology™
SuperArray’s shRNA Design AlgorithmExperimentally verified computer algorithm insures gene-specificity and efficacy.Zhou H, Zeng X, Wang Y and Seyfarth BR. A Three-Phase Algorithm for Computer Aided siRNA Design. Informatica 2006 30: 357-364.Insures Efficacy by including filters for many of the chemical and sequence properties of shRNA known to be important for activity.
LengthGC ContentThermostability bias at 5’-end of antisense strandAvoiding tandem repeats and palidromes
Insures Specificity with Smith-Waterman sequence alignment algorithm, “Better than BLAST”Effective (>70%) knock down determined by rigorous real-time qRT-PCR assay. GUARANTEED!
Pathway-Centric Tools and Technology™
SureSilencing shRNA Validation ProcessTriplicate transfections of negative control shRNA and each of four shRNA designs per gene (HEK 293T)After 48 hours, isolate total RNATriplicate real-time RT-PCR characterization of gene of interest (GOI) and housekeeping gene (HKG) for each of the five triplicate transfectionsCalculate average percent knockdown and 95% confidence intervalAssesses reliability of results by determining whether they are distinguishable from other lower levels of knockdown
Pathway-Centric Tools and Technology™
SureSilencing shRNA Validation ProcessError Model Calculates:
Comprehensive propagation of errors determining total overall experimental variationObserved Knockdown = average decrease in gene expressionImplicated Knockdown = 95 % confidence interval about that mean
Accounts for:Transfection EfficiencyPCR ReproducibilityBiological Sample ConsistencyPCR Amplification Efficiency
White Paper:“Did Your RNAi Experiment Work?! Reliably Validating RNA Interference with Real-Time PCR”http://www.superarray.com/manuals/shRNAwhitepaper.pdf
Pathway-Centric Tools and Technology™
SureSilencing shRNA Validation
Successful Design:KD > 70.0 % and lower 95 % C.I. extreme > 55.5 %Failed Design:KD < 33.3 % and higher 95 % C.I. extreme < 55.5 %Successful Gene = at least two out four designs Successful
Pathway-Centric Tools and Technology™
SureSilencing shRNA Validation Results:Successful Designs
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Pathway-Centric Tools and Technology™
SureSilencing shRNA Validation Results:Failed Designs
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Pathway-Centric Tools and Technology™
SureSilencing shRNA Validation Results
329 Designs Tested, 221 Successful Designs or 67.2 %Original publication by The RNAi Consortium (TRC) reports only
~ 31 to 38 % success rate using the same definition of success
329 tested designs represent 86 Genes Tested2 out 4 designs successful for 74 genes or 86.0 %Binomial Distribution:
Project to EVERY human, mouse, and rat gene89.33 % genes should have 2 out of 4 successful designs
Two out for Four Successful Designs Per Gene IS an Enforceable Guarantee!
Pathway-Centric Tools and Technology™
Benefits of Vector Based System
SureSilencing shRNA Expression Vector☺ Selection for stably transfected cells
Follow slow responses to suppression at the RNA level
☺ Enrichment for transiently transfected cellsFollow quick responses to suppression at the RNA levelMonitor with fluorescence microscopy-based assays
☺ Identify and track transfected cellsAllows use of more difficult or easier to transfect cellsDetermine transfection efficiency
☺ Renewable: One purchase completes your projectAmplified in transformed bacteria
Pathway-Centric Tools and Technology™
SureSilencing™ shRNA Application ExampleFACS-Based Enrichment for GFP-Expressing Cells
70.8 (68.4, 73.0)52TP53Tumor protein p53
71.8 (69.7, 73.8)37PRKCAProtein Kinase C alpha
Sorted PopulationPre-Sorted PopulationPercent Knockdown
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“Polyclonal” Stably Transfected Cells
Human AHR gene
Initial stably transfected population appears to fail guarantee, but …Random integration sites affect shRNA expression and percent KDAverage KD of all integration sites seen – some better than others
Pathway-Centric Tools and Technology™
Individual Stably Transfected Clones
Human AHR gene
Clone cells stably transfected with two best designs by limited dilutionRe-validate clones: Two out of five tested now successful and so is the design
Pathway-Centric Tools and Technology™
Available SureSilencing shRNA Plasmids
Pre-designed shRNA Plasmids are available forEVERY Human, Mouse, or Rat GeneTo search for your genes of interest and find the catalog numbers of the SureSilencing shRNA, visit our website at:http://www.superarray.com/RNAisearch.php
Pathway-Centric Tools and Technology™
SUMMARYBenefits of Vector Based System
Renewable resource; life-time supply with single purchaseSelect or enrich for pure population of knock down cellsApplicable to virtually any cell line:
Low or High transfection efficienciesExceptions: Primary Cells, Macrophages
Track short-term effects OR assay long-term effects
Algorithm & Validation ProcessStringent design process & rigorous qRT-PCR assayHigh rate of success – twice that reported by TRC
Guaranteed SuccessTwo successful clones delivered with > 70% knockdownControl for non-specific and off-target effects