Pathway-Centric Tools and Technology™

SureSilencing™ shRNA
Technology Overview

Guaranteed Plasmid-Based RNA Interference...
Pathway-Centric Tools and Technology™

Topics to be Covered
Challenges Facing RNA Interference Researchers
Solutions SureS...
Pathway-Centric Tools and Technology™

RNA Interference Challenges
Effectiveness:
Reconciling differences between knockdow...
Pathway-Centric Tools and Technology™

What SureSilencing shRNA Provides
Guaranteed Performance:
Suppress expression of ge...
Pathway-Centric Tools and Technology™

THE SureSilencing shRNA GUARANTEE
At least two of the four provided SureSilencing™
...
Pathway-Centric Tools and Technology™

What is SureSilencing™ shRNA?
Pre-designed shRNA constructs specific for a given ta...
Pathway-Centric Tools and Technology™

SureSilencing shRNA Plasmid Backbones
Puromycin

Neomycin

GFP
Pathway-Centric Tools and Technology™

SureSilencing shRNA Plasmid Backbones
Life-time supply with single purchase
Bacteri...
Pathway-Centric Tools and Technology™

SuperArray’s shRNA Design Algorithm
Experimentally verified computer algorithm insu...
Pathway-Centric Tools and Technology™

SureSilencing shRNA Validation Process
Triplicate transfections of negative control...
Pathway-Centric Tools and Technology™

SureSilencing shRNA Validation Process
Error Model Calculates:
Comprehensive propag...
Pathway-Centric Tools and Technology™

SureSilencing shRNA Validation
Successful Design:
KD > 70.0 % and lower 95 % C.I. e...
Pathway-Centric Tools and Technology™

SureSilencing shRNA Validation Results:
Successful Designs
110
100
90

Observed KD
...
Pathway-Centric Tools and Technology™

SureSilencing shRNA Validation Results:
Failed Designs
60
50
40
30

Observed KD

20...
Pathway-Centric Tools and Technology™

SureSilencing shRNA Validation Results
329 Designs Tested, 221 Successful Designs o...
Pathway-Centric Tools and Technology™

Benefits of Vector Based System
SureSilencing shRNA Expression Vector
☺ Selection f...
Pathway-Centric Tools and Technology™

SureSilencing™ shRNA Application Example
FACS-Based Enrichment for GFP-Expressing C...
Pathway-Centric Tools and Technology™

“Polyclonal” Stably Transfected Cells

Human AHR gene

Initial stably transfected p...
Pathway-Centric Tools and Technology™

Individual Stably Transfected Clones

Human AHR gene

Clone cells stably transfecte...
Pathway-Centric Tools and Technology™

Available SureSilencing shRNA Plasmids
Pre-designed shRNA Plasmids are available fo...
Pathway-Centric Tools and Technology™

SUMMARY
Benefits of Vector Based System
Renewable resource; life-time supply with s...
Pathway-Centric Tools and Technology™

SureSilencing™ shRNA
Technology Overview

Guaranteed Plasmid-Based RNA Interference...
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Sure silencing

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Sure silencing

  1. 1. Pathway-Centric Tools and Technology™ SureSilencing™ shRNA Technology Overview Guaranteed Plasmid-Based RNA Interference For EVERY Human, Mouse, and Rat Gene
  2. 2. Pathway-Centric Tools and Technology™ Topics to be Covered Challenges Facing RNA Interference Researchers Solutions SureSilencing shRNA Provides How SureSilencing shRNA Works Vector, Design Algorithm, Validation Process Guaranteed Success How YOU Can Use SureSilencing shRNA
  3. 3. Pathway-Centric Tools and Technology™ RNA Interference Challenges Effectiveness: 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.
  4. 4. Pathway-Centric Tools and Technology™ What SureSilencing shRNA Provides Guaranteed 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.
  5. 5. 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.
  6. 6. Pathway-Centric Tools and Technology™ What is SureSilencing™ shRNA? Pre-designed shRNA constructs specific for a given target gene Four (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 genomes All separately cloned into a mammalian expression vector system with the same selectable marker
  7. 7. Pathway-Centric Tools and Technology™ SureSilencing shRNA Plasmid Backbones Puromycin Neomycin GFP
  8. 8. Pathway-Centric Tools and Technology™ SureSilencing shRNA Plasmid Backbones Life-time supply with single purchase Bacterial origin of replication and ampicillin-resistance marker Choice of one of three mammalian markers Neomycin for stable transfections Puromycin alternative marker for stable transfections GFP for transient transfections Minimize non-specific off-target and toxic side effects U1 promoter, transcribed by RNA Polymerase II, that normally transcribes mRNA Provides moderate shRNA expression level Other promoter system express too much shRNA
  9. 9. Pathway-Centric Tools and Technology™ SuperArray’s shRNA Design Algorithm Experimentally verified computer algorithm insures genespecificity 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. Length GC Content Thermostability bias at 5’-end of antisense strand Avoiding 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!
  10. 10. Pathway-Centric Tools and Technology™ SureSilencing shRNA Validation Process Triplicate transfections of negative control shRNA and each of four shRNA designs per gene (HEK 293T) After 48 hours, isolate total RNA Triplicate real-time RT-PCR characterization of gene of interest (GOI) and housekeeping gene (HKG) for each of the five triplicate transfections Calculate average percent knockdown and 95% confidence interval Assesses reliability of results by determining whether they are distinguishable from other lower levels of knockdown
  11. 11. Pathway-Centric Tools and Technology™ SureSilencing shRNA Validation Process Error Model Calculates: Comprehensive propagation of errors determining total overall experimental variation Observed Knockdown = average decrease in gene expression Implicated Knockdown = 95 % confidence interval about that mean Accounts for: Transfection Efficiency PCR Reproducibility Biological Sample Consistency PCR Amplification Efficiency White Paper: “Did Your RNAi Experiment Work?! Reliably Validating RNA Interference with Real-Time PCR” http://www.superarray.com/manuals/shRNAwhitepaper.pdf
  12. 12. 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
  13. 13. Pathway-Centric Tools and Technology™ SureSilencing shRNA Validation Results: Successful Designs 110 100 90 Observed KD 80 70 60 50 40 30 20 10 0 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 Rank Order 81 86 91 96 101 106 111 116 121
  14. 14. Pathway-Centric Tools and Technology™ SureSilencing shRNA Validation Results: Failed Designs 60 50 40 30 Observed KD 20 10 0 1 2 3 4 5 6 7 8 -10 -20 -30 -40 -50 -60 Rank Order 9 10 11
  15. 15. 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 Tested 2 out 4 designs successful for 74 genes or 86.0 % Binomial Distribution: Project to EVERY human, mouse, and rat gene 89.33 % genes should have 2 out of 4 successful designs Two out for Four Successful Designs Per Gene IS an Enforceable Guarantee!
  16. 16. 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 cells Follow quick responses to suppression at the RNA level Monitor with fluorescence microscopy-based assays ☺ Identify and track transfected cells Allows use of more difficult or easier to transfect cells Determine transfection efficiency ☺ Renewable: One purchase completes your project Amplified in transformed bacteria
  17. 17. Pathway-Centric Tools and Technology™ SureSilencing™ shRNA Application Example FACS-Based Enrichment for GFP-Expressing Cells Percent Knockdown Pre-Sorted Population Sorted Population PRKCA Protein Kinase C alpha 37 71.8 (69.7, 73.8) TP53 Tumor protein p53 52 70.8 (68.4, 73.0)
  18. 18. Pathway-Centric Tools and Technology™ “Polyclonal” Stably Transfected Cells Human AHR gene Initial stably transfected population appears to fail guarantee, but … Random integration sites affect shRNA expression and percent KD Average KD of all integration sites seen – some better than others
  19. 19. Pathway-Centric Tools and Technology™ Individual Stably Transfected Clones Human AHR gene Clone cells stably transfected with two best designs by limited dilution Re-validate clones: Two out of five tested now successful and so is the design
  20. 20. Pathway-Centric Tools and Technology™ Available SureSilencing shRNA Plasmids Pre-designed shRNA Plasmids are available for EVERY Human, Mouse, or Rat Gene To 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
  21. 21. Pathway-Centric Tools and Technology™ SUMMARY Benefits of Vector Based System Renewable resource; life-time supply with single purchase Select or enrich for pure population of knock down cells Applicable to virtually any cell line: Low or High transfection efficiencies Exceptions: Primary Cells, Macrophages Track short-term effects OR assay long-term effects Algorithm & Validation Process Stringent design process & rigorous qRT-PCR assay High rate of success – twice that reported by TRC Guaranteed Success Two successful clones delivered with > 70% knockdown Control for non-specific and off-target effects
  22. 22. Pathway-Centric Tools and Technology™ SureSilencing™ shRNA Technology Overview Guaranteed Plasmid-Based RNA Interference For EVERY Human, Mouse, and Rat Gene

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