sure silencing

Pathway-Centric Tools and Technology™

SureSilencing™ shRNATechnology Overview

Guaranteed Plasmid-Based RNA InterferenceFor EVERY Human, Mouse, and Rat Gene


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


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.


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.


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.


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


SureSilencing shRNA Plasmid Backbones

Neomycin Puromycin

GFP


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


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!


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


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


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


SureSilencing shRNA Validation Results:Successful Designs

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SureSilencing shRNA Validation Results:Failed Designs

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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!


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


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


“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


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


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


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


SureSilencing™ shRNATechnology Overview

Guaranteed Plasmid-Based RNA InterferenceFor EVERY Human, Mouse, and Rat Gene

sure silencing

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