Download Planning and Executing siRNA Experiments—Good Practices for

Planning and Executing siRNA Experiments—Good Practices
for Optimal Results
Garre% Re(g, PhD
Abstract
Func%onal analysis by mRNA knockdown using siRNAs is now rou%ne in many molecular biology labs. However, many RNAi-­‐related experiments fail due to diversion from simple, good prac%ces. This webinar will review the steps leading to successful siRNA experiments, including: •  Understanding the target transcript •  siRNA selec%on •  Choosing the cell type •  Valida%ng the assay •  Including appropriate biological controls 2
DsiRNA—Intracellular Pathway
3
DsiRNA Processing
4
DsiRNA Processing
5
RNAi-Mediated Knockdown or Artifact? Amplification Plot
qPCR – Gene of Interest (GOI) Expression in HeLa Cells
∆ Rn
∆ Cq > 3.3, 90% knockdown
Untreated controls
siRNA targeting gene of
interest
Cycle
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Strategy
Iden%fy target gene of interest DsiRNA selec%on Cell line selec%on Op%mize experimental condi%ons Controlled pilot experiment 7
Op%mized experiment: gene of interest knockdown Understanding the Transcript
2° Structure Iden%fy target gene of interest Transcript variants Remaining mRNA Levels (%) Species varia%on GOI Knockdown in HeLa Cells at 1 nM Normalized to Hs HPRT and SFRS9 vs NC1, NC5, and NC7 120 110 100 90 80 70 60 50 40 30 20 10 0 0 500 1000 1500 2000 2500 3000 3500 4000 4500 siRNA (Hs LocaFons) 8
Hs STAT3 574-­‐720 (FAM) Hs STAT3 3904-­‐4036 (MAX) INTEGRATED DNA TECHNOLOGIES
5' UTR CDS 3' UTR 5000 qPCR Assay Discordance
2° Structure Iden%fy target gene of interest Transcript variants Species varia%on 120 GOI Knockdown in HeLa Cells at 1 nM Normalized to Hs HPRT and SFRS9 vs NC1, NC5, and NC7 110 Assay discordance appears at the 3’-­‐end of the transcript. Measured mRNA levels show significant divergence Retained mRNA fragments “Geographically” spaced qPCR assays mRNA Remaining (%) 100 90 80 70 60 50 40 30 20 10 0 0 9
500 Assay 1 1000 Assay 2 5' UTR CDS 1500 3' UTR Understanding the Transcript
2° Structure Iden%fy target gene of interest Transcript variants Species varia%on qPCR Assay Loc
DsiRNA Loc
Transcript variants (and rela%ve abundance) can affect results in a qPCR assay and DsiRNA loca%on-­‐
dependent fashion. hZp://www.informaFcs.jax.org/genes.shtml 10
Understanding the Transcript
2° Structure Iden%fy target gene of interest Transcript variants Species varia%on Hs GOI
Mm GOI
Region of Mm/Hs sequence homology
Interspecies alignment of mRNA sequence can affect future experimental direc%ons. 11
Selecting an Effective siRNA
DsiRNA selec%on Design rules Design tools 12
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
Selecting an Effective siRNA
DsiRNA selec%on Tuschl
Design rules Design tools 13
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 3ʹ′ end of the sense siRNA to TT
4.  Or search for NARN17YNN
5.  Target sequence should have a GC content of around 50%
Selecting an Effective siRNA
DsiRNA selec%on Ui-Tei
Design rules Design tools 14
Nucleic Acids Res (2004) 32(3):936-48
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
Selecting an Effective siRNA
DsiRNA selec%on Design rules Design tools 15
Selecting an Effective siRNA
DsiRNA selec%on Design rules Design tools 16
Selecting an Effective siRNA
DsiRNA selec%on Design rules Design tools 17
Guarantee: 2 of the top 3 ranked DsiRNAs will exhibit >70% knockdown at 10 nM transfecFon 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. Cell Line
Expression profile Cell line selec%on Literature search Assay valida%on Relative Abundance
Hs GAPDH Tissue Prevalence
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http://biogps.org/#goto=welcome
INTEGRATED DNA TECHNOLOGIES
Cell Line
Expression profile Cell line selec%on Literature search Assay valida%on Biomaterials 33 (2012) 1154-1161
GAPDH NIH 3T3 murine fibroblasts 12,500 cells/cm2 6.25 – 50 nM qPCR 19
Cell Line
Expression Profile Cell line selec%on Literature Search Assay valida%on § 
qPCR § 
Western § 
bDNA Northern § 
§ 
Phenotype Amplification Plot
∆ Rn
qPCR – Gene of Interest Expression in Candidate Cell Line
Untreated controls
106 Cycle
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INTEGRATED DNA TECHNOLOGIES
105 104 103 102 101 Cell Line
Expression Profile Cell line selec%on Literature Search Assay valida%on 21
INTEGRATED DNA TECHNOLOGIES
Cell Line
Expression Profile Cell line selec%on Literature Search Assay valida%on HPRT mRNA and Protein Knockdown
10nM Transfection in HeLa Cells
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Optimizing Conditions
Transfec%on Controls Op%mize experimental condi%ons OpFmizing U87 Cell TransfecFons HPRT Knockdown Normalized to SFRS9 24hr Reverse TransfecFons 110% 100% Remaining mRNA Levels (%) 90% 80% 70% 6uL INTERFERin 60% 3uL TKO 50% 1uL siLentFect 40% 2uL RNAiMAX 30% 20% 10% 0% NC1 10nM 23
HPRT 10nM Optimizing Conditions
Transfec%on Controls/Variables Op%mize experimental condi%ons Nega%ve Control DsiRNAs 5'3'5'3'5'3'-
CGUUAAUCGCGUAUAAUACGCGUAT
|||||||||||||||||||||||||
CAGCAAUUAGCGCAUAUUAUGCGCAUA
CAUAUUGCGCGUAUAGUCGCGUUAG
|||||||||||||||||||||||||
UGGUAUAACGCGCAUAUCAGCGCAAUC
GGCGCGUAUAGUCGCGCGUAUAGTC
|||||||||||||||||||||||||
CUCCGCGCAUAUCAGCGCGCAUAUCAG
Addi%onal parameters to op%mize: TransfecGon reagent
Dose-­‐response -­‐ reagent
Cell seeding density
Dose-­‐response – DsiRNA
Forward/reverse
Time course
Reagent:DsiRNA raGo
Posi%ve Control DsiRNA – HPRT (Hypoxanthine-­‐guanine phosphoribosyltransferase) 5'3'-
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GCCAGACUUUGUUGGAUUUGAAATT
|||||||||||||||||||||||||
UUCGGUCUGAAACAACCUAAACUUUAA
Experimental Setup
Cells Only
HPRT Pos – 10 nM
Reagent Only
HPRT Pos – 1 nM
Neg siRNA#1 – 10 nM
HPRT Pos – 0.1 nM
Neg siRNA#2 – 10 nM
GOI siRNA – 10 nM
•  Nega%ve controls •  Posi%ve controls •  DsiRNA targe%ng gene of interest •  Biological replicates •  Technical replicates 25
Summary
2° Structure Iden%fy target gene of interest Transcript variants DsiRNA selec%on Species varia%on Design rules Design tools Transfec%on Controls Op%mize experimental condi%ons Expression profile Cell line selec%on Assay valida%on Controlled pilot experiment 26
Literature search Op%mized experiment: gene of interest knockdown Additional Resources
EducaFonal Resources at www.IDTDNA.com Under Support & Educa0on Menu •  DECODED Newsleeer (www.IDTDNA.com/
DECODED) •  Video Library •  Frequently Asked Ques%ons •  More… Design Tools at www.IDTDNA.com/SciTools or Under the Tools Menu •  Custom RNAi Design Tool •  Predesigned DsiRNA Selec%on Tool •  PrimeTime® qPCR Assays Tool •  PrimerQuest® Tool for PCR and qPCR Design Customer Care and Technical Support for Design, Experimental Issues, and Ordering Help •  [email protected] 27
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Additional Resources
AddiFonal Product InformaFon: •  More informa%on on DsiRNA 27mer duplexes at www.idtdna.com , under Products &Services/DsiRNA •  More informa%on on PrimeTime® qPCR Assays and products at www.IDTDNA.com/PrimeTime Related IDT PublicaFons Molecular Therapy (2012) 20(3):483-­‐512. Gene Therapy (2011) 18:1111-­‐1120. OligonucleoAdes (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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Integrated DNA Technologies, Inc. 1710 Commercial Park Coralville, Iowa 52241 USA