This document provides guidance on planning and executing successful siRNA experiments through following good practices. It reviews key steps including understanding the target transcript through identifying variants and structures, selecting effective siRNAs using design tools and rules, choosing a cell line with appropriate expression profiles, optimizing experimental conditions through controls and pilot experiments, and validating the assay. Following these steps can help achieve optimized gene knockdown.

1. Planning and Executing siRNA Experiments—Good Practices
for Optimal Results
Garrett Rettig, PhD

2. 2
Abstract
Functional analysis by mRNA knockdown using siRNAs is now routine in
many molecular biology labs. However, many RNAi-related experiments fail
due to diversion from simple, good practices. This webinar will review the
steps leading to successful siRNA experiments, including:
• Understanding the target transcript
• siRNA selection
• Choosing the cell type
• Validating the assay
• Including appropriate biological controls

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DsiRNA—Intracellular Pathway

4. 4
DsiRNA Processing

5. 5
DsiRNA Processing

6. 6
RNAi-Mediated Knockdown or Artifact?
Untreated controls
siRNA targeting gene of
interest
Cycle
∆Rn
∆ Cq > 3.3, 90% knockdown
Amplification Plot
qPCR – Gene of Interest (GOI) Expression in HeLa Cells

7. 7
Strategy
Optimized experiment:
gene of interest
knockdown
Identify target gene of interest
DsiRNA selection
Cell line selection
Optimize experimental conditions
Controlled pilot experiment

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INTEGRATED DNA TECHNOLOGIES
Understanding the Transcript
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0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000
RemainingmRNALevels(%)
siRNA (Hs Locations)
GOI Knockdown in HeLa Cells at 1 nM
Normalized to Hs HPRT and SFRS9 vs NC1, NC5, and NC7
Hs STAT3 574-720 (FAM) Hs STAT3 3904-4036 (MAX) 5' UTR CDS 3' UTR
Identify target
gene of interest
2° Structure
Transcript
variants
Species
variation

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qPCR Assay Discordance
Identify target
gene of interest
2° Structure
Transcript
variants
Species
variation
Assay discordance appears at the
3’-end of the transcript.
Measured mRNA levels show
significant divergence
Retained mRNA fragments
“Geographically” spaced qPCR
assays
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mRNARemaining(%)
GOI Knockdown in HeLa Cells at 1 nM
Normalized to Hs HPRT and SFRS9 vs NC1, NC5, and NC7
Assay 1 Assay 2 5' UTR CDS 3' UTR

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Understanding the Transcript
http://www.informatics.jax.org/genes.shtml
qPCR Assay Loc DsiRNA Loc
Identify target
gene of interest
2° Structure
Transcript
variants
Species
variation
Transcript variants (and relative abundance) can affect results in a qPCR assay and DsiRNA location-
dependent fashion.
1
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3
4
5

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Understanding the Transcript
Identify target
gene of interest
2° Structure
Transcript
variants
Species
variation
Hs GOI
Mm GOI
Region of Mm/Hs sequence homology
Interspecies alignment of mRNA sequence can affect future experimental directions.

12. 12
Selecting an Effective siRNA
Reynolds Nat Biotechnol (2004) 22(3):326-30
1. siRNA targeted sequence is usually 21 nt in length
2. Avoid regions within 50100 bp of the start codon and the termination codon
3. Avoid intron regions
4. Avoid stretches of 4 or more bases (AAAA, CCCC)
5. Avoid regions with GC content <30% or >60%
6. Avoid repeats and low complexity sequence
7. Avoid SNP sites
8. Perform BLAST homology search to avoid off-target effects on other genes or sequences
9. Design negative controls as scrambled sequence of the target
DsiRNA selection
Design rules
Design tools

13. 13
Selecting an Effective siRNA
Tuschl Methods (2002) 26(2):199-213
1. Select targeted region from a given cDNA sequence 50-100 nt downstream of start codon
2. First search for 21-nt sequence motif AAN19. If no suitable sequence found, then,
3. Search for 23-nt sequence motif NAN21 and convert the 3 end of the sense siRNA to TT
4. Or search for NARN17YNN
5. Target sequence should have a GC content of around 50%
DsiRNA selection
Design rules
Design tools
DsiRNA selection

14. 14
Selecting an Effective siRNA
1. A/U at the 5' end of the antisense strand
2. G/C at the 5' end of the sense strand
3. At least five A/U residues in the 5' terminal one-third of the antisense strand
4. The absence of any GC stretch of more than 9 nt in length
Ui-Tei Nucleic Acids Res (2004) 32(3):936-48
DsiRNA selection
Design rules
Design tools

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Selecting an Effective siRNA
DsiRNA selection
Design rules
Design tools

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Selecting an Effective siRNA
DsiRNA selection
Design rules
Design tools

17. 17
Selecting an Effective siRNA
DsiRNA selection
Design rules
Design tools
Guarantee: 2 of the top 3 ranked DsiRNAs will
exhibit >70% knockdown at 10 nM transfection
in a well-controlled experiment
Tested 50 genes to confirm the frequency of
achieving guaranteed knockdown.
• 42/50 genes had 2 out of the first 3 ranked
DsiRNAs pass at 10 nM.
• 50/50 genes had at least 3 passing DsiRNAs
out of the tested set of 10 at 10 nM.

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INTEGRATED DNA TECHNOLOGIES
Cell Line
Expression profile
Cell line selection Literature search
Assay validation
http://biogps.org/#goto=welcome
Hs GAPDH Tissue Prevalence
RelativeAbundance

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Cell Line
Biomaterials 33 (2012) 1154-1161
Expression profile
Cell line selection Literature search
Assay validation
GAPDH
NIH 3T3 murine fibroblasts
12,500 cells/cm2
6.25 – 50 nM
qPCR

20. 20
INTEGRATED DNA TECHNOLOGIES
Expression Profile
Cell line selection Literature Search
Assay validation
Cell Line
Untreated controls
106 105 104 103
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Amplification Plot
qPCR – Gene of Interest Expression in Candidate Cell Line∆Rn
Cycle
 Western  bDNA  Phenotype qPCR  Northern

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INTEGRATED DNA TECHNOLOGIES
Cell Line
Expression Profile
Cell line selection Literature Search
Assay validation

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INTEGRATED DNA TECHNOLOGIES
Cell Line
Expression Profile
Cell line selection Literature Search
Assay validation HPRT mRNA and Protein Knockdown
10nM Transfection in HeLa Cells

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10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
110%
NC1 10nM HPRT 10nM
RemainingmRNALevels(%)
Optimizing U87 Cell Transfections
HPRT Knockdown Normalized to SFRS9
24hr Reverse Transfections
6uL INTERFERin
3uL TKO
1uL siLentFect
2uL RNAiMAX
Optimize
experimental
conditions
Transfection
Controls
Optimizing Conditions

24. 24
Optimizing Conditions
Positive Control DsiRNA – HPRT
(Hypoxanthine-guanine phosphoribosyltransferase)
Optimize
experimental
conditions
Transfection
Controls/Variables
5'- CGUUAAUCGCGUAUAAUACGCGUAT
|||||||||||||||||||||||||
3'- CAGCAAUUAGCGCAUAUUAUGCGCAUA
5'- CAUAUUGCGCGUAUAGUCGCGUUAG
|||||||||||||||||||||||||
3'- UGGUAUAACGCGCAUAUCAGCGCAAUC
5'- GGCGCGUAUAGUCGCGCGUAUAGTC
|||||||||||||||||||||||||
3'- CUCCGCGCAUAUCAGCGCGCAUAUCAG
5'- GCCAGACUUUGUUGGAUUUGAAATT
|||||||||||||||||||||||||
3'- UUCGGUCUGAAACAACCUAAACUUUAA
Negative Control DsiRNAs
Additional parameters to optimize:
Transfection reagent
Dose-response - reagent
Cell seeding density
Dose-response – DsiRNA
Forward/reverse
Time course
Reagent:DsiRNA ratio

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Experimental Setup
• Negative controls
• Positive controls
• DsiRNA targeting gene of interest
• Biological replicates
• Technical replicates
Cells Only
Reagent Only
Neg siRNA#1 – 10 nM
HPRT Pos – 10 nM
HPRT Pos – 1 nM
Neg siRNA#2 – 10 nM
HPRT Pos – 0.1 nM
GOI siRNA – 10 nM

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Summary
Optimized experiment:
gene of interest
knockdown
Identify target
gene of interest
2° Structure
Transcript
variants
Species
variation
DsiRNA selection
Design rules
Design tools
Cell line selection
Optimize
experimental
conditions
Controlled pilot
experiment
Expression profile
Literature search
Assay validation
Transfection
Controls

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INTEGRATED DNA TECHNOLOGIES
Additional Resources
Educational Resources at www.IDTDNA.com Under
Support & Education Menu
• DECODED Newsletter
(www.IDTDNA.com/DECODED)
• Video Library
• Frequently Asked Questions
• More…
Design Tools at www.IDTDNA.com/SciTools or
Under the Tools Menu
• Custom RNAi Design Tool
• Predesigned DsiRNA Selection Tool
• PrimeTime® qPCR Assays Tool
• PrimerQuest® Tool for PCR and qPCR Design
Customer Care and Technical Support for Design,
Experimental Issues, and Ordering Help
• custcare@IDTDNA.com

28. 28
INTEGRATED DNA TECHNOLOGIES
Additional Resources
Additional Product Information:
• More information on DsiRNA 27mer duplexes at
www.idtdna.com , under Products &Services/DsiRNA
• More information on PrimeTime® qPCR Assays and
products at www.IDTDNA.com/PrimeTime
Related IDT Publications
• Molecular Therapy (2012) 20(3):483-512.
• Gene Therapy (2011) 18:1111-1120.
• Oligonucleotides (2008) 18:305-320.
• Curr Opin in Mol Ther (2007) 9(2):110-118.
• Nature Methods (2006) Online 23 August;
DOI:10.1038.
• Nucleic Acids Research (2005) 33:4140-4156.
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