1. Pathway-Centric Tools and Technology™
SureSilencing™ shRNA
Technology Overview
Guaranteed Plasmid-Based RNA Interference
For EVERY Human, Mouse, and Rat Gene
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. 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. 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. 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. 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
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. 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. 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. 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. 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
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. 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. 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. 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. 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. 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. 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. Pathway-Centric Tools and Technology™
SureSilencing™ shRNA
Technology Overview
Guaranteed Plasmid-Based RNA Interference
For EVERY Human, Mouse, and Rat Gene
