Planning and Executing siRNA Experiments—Good Practices for Optimal Results

Planning and Executing siRNA Experiments—Good Practices for Optimal Results

Garrett Rettig, PhD

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 transcriptsiRNA selectionChoosing the cell type Validating the assayIncluding appropriate biological controls

3

DsiRNA—Intracellular Pathway

4

DsiRNA Processing

5

DsiRNA Processing

6

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

8INTEGRATED DNA TECHNOLOGIES

Understanding the Transcript

0 500 1000 1500 2000 2500 3000 3500 4000 4500 50000

102030405060708090

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

(%)


2° Structure

Transcript variants

Species variation

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qPCR Assay Discordance


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

10

20

30

40

50

60

70

80

90

100

110

120

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

(%)

10


http://www.informatics.jax.org/genes.shtml

qPCR Assay Loc DsiRNA Loc


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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2

3

4

5

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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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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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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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DsiRNA selection

Design rules

Design tools

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DsiRNA selection

Design rules

Design tools

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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.


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


Expression Profile

Cell line selection Literature Search

Assay validation

Cell Line

Untreated controls

106 105 104 103 102 101

Amplification PlotqPCR – Gene of Interest Expression in Candidate Cell Line

∆ R

n

Cycle

Western bDNA Phenotype qPCR Northern


Cell Line

Expression Profile


Assay validation


Cell Line

Expression Profile


Assay validation HPRT mRNA and Protein Knockdown10nM Transfection in HeLa Cells

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


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


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

• [email protected]

http://www.idtdna.com/

http://www.idtdna.com/SciTools

mailto:[email protected]


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.

Integrated DNA Technologies, Inc.1710 Commercial ParkCoralville, Iowa 52241USA

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Date post:	14-Jun-2015
Category:	Science
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Planning and Executing siRNA Experiments—Good Practices for Optimal Results

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