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Planning and Executing siRNA Experiments—Good Practices for Optimal Results
Garrett Rettig, PhD
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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 transcriptsiRNA selectionChoosing the cell type Validating the assayIncluding appropriate biological controls
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DsiRNA—Intracellular Pathway
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DsiRNA Processing
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DsiRNA Processing
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RNAi-Mediated Knockdown or Artifact?
Untreated controls
siRNA targeting gene of interest
Cycle
∆ R
n
∆ Cq > 3.3, 90% knockdown
Amplification PlotqPCR – Gene of Interest (GOI) Expression in HeLa Cells
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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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8INTEGRATED DNA TECHNOLOGIES
Understanding the Transcript
0 500 1000 1500 2000 2500 3000 3500 4000 4500 50000
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100110120
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' UTRCDS 3' UTR
siRNA (Hs Locations)
Rem
aini
ng m
RNA
Lev
els
(%)
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
0 500 1000 15000
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30
40
50
60
70
80
90
100
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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
mRN
A R
emai
ning
(%)
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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.
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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.
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Selecting an Effective siRNA
Reynolds Nat Biotechnol (2004) 22(3):326-30
1. siRNA targeted sequence is usually 21 nt in length2. Avoid regions within 50100 bp of the start codon and the termination codon3. Avoid intron regions4. Avoid stretches of 4 or more bases (AAAA, CCCC)5. Avoid regions with GC content <30% or >60%6. Avoid repeats and low complexity sequence7. Avoid SNP sites8. Perform BLAST homology search to avoid off-target effects on other genes or sequences9. Design negative controls as scrambled sequence of the target
DsiRNA selection
Design rules
Design tools
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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 codon2. 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 TT4. Or search for NARN17YNN5. Target sequence should have a GC content of around 50%
DsiRNA selection
Design rules
Design tools
DsiRNA selection
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Selecting an Effective siRNA
1. A/U at the 5' end of the antisense strand2. G/C at the 5' end of the sense strand3. At least five A/U residues in the 5' terminal one-third of the antisense strand4. 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
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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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18INTEGRATED DNA TECHNOLOGIES
Cell Line
Expression profile
Cell line selection Literature search
Assay validation
http://biogps.org/#goto=welcome
Hs GAPDH Tissue Prevalence
Rel
ativ
e A
bund
ance
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Cell Line
Biomaterials 33 (2012) 1154-1161
Expression profile
Cell line selection Literature search
Assay validation
GAPDHNIH 3T3 murine fibroblasts
12,500 cells/cm2
6.25 – 50 nMqPCR
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20INTEGRATED DNA TECHNOLOGIES
Expression Profile
Cell line selection Literature Search
Assay validation
Cell Line
Untreated controls
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Amplification PlotqPCR – Gene of Interest Expression in Candidate Cell Line
∆ R
n
Cycle
Western bDNA Phenotype qPCR Northern
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21INTEGRATED DNA TECHNOLOGIES
Cell Line
Expression Profile
Cell line selection Literature Search
Assay validation
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22INTEGRATED DNA TECHNOLOGIES
Cell Line
Expression Profile
Cell line selection Literature Search
Assay validation HPRT mRNA and Protein Knockdown10nM Transfection in HeLa Cells
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23NC1 10nM HPRT 10nM
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
110%
Optimizing U87 Cell Transfections HPRT Knockdown Normalized to SFRS9
24hr Reverse Transfections
6uL INTERFERin3uL TKO1uL siLentFect2uL RNAiMAX
Rem
aini
ng m
RNA
Lev
els
(%)
Optimize experimental
conditions
Transfection
Controls
Optimizing Conditions
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Optimizing Conditions
Positive Control DsiRNA – HPRT(Hypoxanthine-guanine phosphoribosyltransferase)
Optimize experimental
conditions
Transfection
Controls/Variables5'- CGUUAAUCGCGUAUAAUACGCGUAT |||||||||||||||||||||||||3'- CAGCAAUUAGCGCAUAUUAUGCGCAUA
5'- CAUAUUGCGCGUAUAGUCGCGUUAG |||||||||||||||||||||||||3'- UGGUAUAACGCGCAUAUCAGCGCAAUC
5'- GGCGCGUAUAGUCGCGCGUAUAGTC |||||||||||||||||||||||||3'- CUCCGCGCAUAUCAGCGCGCAUAUCAG
5'- GCCAGACUUUGUUGGAUUUGAAATT |||||||||||||||||||||||||3'- UUCGGUCUGAAACAACCUAAACUUUAA
Negative Control DsiRNAs
Additional parameters to optimize:Transfection reagentDose-response - reagentCell seeding densityDose-response – DsiRNAForward/reverseTime courseReagent: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 selectionOptimize
experimental conditions
Controlled pilot experiment
Expression profile
Literature search
Assay validation
Transfection
Controls
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27INTEGRATED DNA TECHNOLOGIES
Additional ResourcesEducational 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
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28INTEGRATED DNA TECHNOLOGIES
Additional ResourcesAdditional 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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