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RNAi: Five Tips to Better Silencing

Anxious to get going with RNAi? These tips will make your next knockdown a knockout

By | January 1, 2006

Thanks to RNA interference it's easier than ever to perform genetic knockouts in cultured cells and live animals. The idea is simple enough: deliver a short interfering RNA (siRNA) for the desired mRNA, wait a few days, and see what happens. In theory, the siRNAs (short double-stranded RNAs homologous to a region of an mRNA) will program the RNA-induced silencing complex to target the desired transcript for degradation.

In practice, however, it takes time, and a lot of optimization, to get the process working. This is less true for researchers whose target genes correspond to precloned interference constructs such as those available from Cold Spring Harbor Laboratory (http://codex.cshl.edu) or commercial vendors such as Sigma-Aldrich, Open Biosystems, and System Biosciences. You might also get lucky and be able to purchase ready-to-use siRNAs from Ambion, Qiagen, or Dharmacon. But suppose you need to go from gene to gene knockout completely from scratch? Don't make a move before reading these five tips.

1. MAKE SURE YOU HAVE THE RIGHT SEQUENCE

Because RNA interference (RNAi) can vary greatly, most suppliers recommend screening four or five siRNAs per transcript. In a 2004 report, siRNA vendor Dharmacon identified eight criteria for optimal inhibitor design.1 In general, siRNAs should be 19 to 21 nucleotides long, lack internal repeats or a high degree of secondary structure, have low (under 50%) G/C content, and lack sequence homology to off-target genes. Thermodynamic stability is also key. The study showed enhanced potency in siRNAs with low internal stability at the sense strand 3' end.

A number of free siRNA-design programs are available online from commercial RNAi tool vendors and academic­ labs.

2. START WITH NAKED siRNA

If you're just starting out, it makes sense to use "naked" siRNAs (i.e., purified double- stranded RNAs) that are delivered by transient transfection. "siRNAs make a lot of sense to use in the instances where you want a very transient decrease in expression to either test a particular hypothesis or ? to evaluate the role of a given sequence as a therapeutic," says Beverly Davidson, a neuroscientist at the University of Iowa, who uses RNAi to knock down genes involved in neurogenetic diseases.

The fastest (albeit more expensive) approach is to order siRNAs custom-made from an oligo supplier. The major RNAi companies sell annealed siRNAs in several purity formats ranging from standard (for in vitro work) to HPLC or PAGE-purified (for in vivo work). You can also make your own siRNA; commercial kits can transcribe two complementary siRNA strands in vitro for subsequent annealing and transfection. (See tables below)

3. MAKE THE RIGHT MODIFICATIONS

Some researchers chemically modify their siRNAs for greater stability or tissue targeting. Chandra Vargeese, vice president of chemistry at San Francisco-based Sirna Therapeutics, says the company's scientists have developed a portfolio of chemical modifications to 2'-OH groups to facilitate local and systemic siRNA delivery, and in some cases even modulate immunostimulatory side effects. "We can go from no modifications to fully modified siRNAs, and anything in between," she says.

Recently the company demonstrated that replacement of ribose sugar 2'-hydroxyl groups with 2'-fluorine, 2'-O-methyl, and 2'-deoxy sugars, and the use of backbone phosphorothioate linkages and 5' and 3' inverted abasic end caps, produced a thousand- fold enhancement in stability in human serum.2 Modifications can significantly increase the cost of your oligos, however. Qiagen, for instance, sells cholesterol- modified RNAs for roughly twice the cost of purified, nonmodified siRNAs, according to company spokesperson Eric Lader.

4. OPTIMIZE SILENCING WITH PLASMIDS

Longer-lasting effects can also be achieved by expressing plasmid-encoded short hairpin RNAs (shRNAs) or microRNAs­ from pol III or pol II promoter sequences, respectively, in vivo or in culture.3,4 shRNAs typically contain stem structures between 19 and 29 nucleotides in length, capped with a five-to-nine base loop sequence; microRNAs are longer, more complicated structures. Because they are transcribed using Pol II, microRNA-based constructs can be expressed conditionally (e.g., using a regulatable promoter), allowing for more sophisticated knockdown experiments. Ultimately, though, both shRNAs and microRNAs are processed in vivo into double-stranded products capable of programming the RISC.5

5. USE VIRUSES FOR IN VIVO WORK

Naked siRNA and shRNA expressed by a standard plasmid vector are fine for screening in cultured cells, but they don't work well in live animals. But viral vectors do. Says Davidson, "If you put naked DNA into a lung or brain you may get very little of the RNAi expressed, but you can use a virus to deliver the payload to your cells of interest or whatever organ you're targeting."

Researchers who use viral vectors ? both adenoviral and lentiviral systems are available ? can employ selectable markers to look for cells in which shRNA has been expressed, and then follow up with an assay for the desired phenotype, says David Root of the Broad Institute at the Massachusetts Institute of Technology. The RNAi effect can then be turned on or off with a conditional expression system, such as the doxycycline- or tetracycline-regulated systems from Clontech, Stratagene, and Invitrogen, among others. (see Table 2 below)

aconstans@the-scientist.com

References

1. A. Reynolds et al., "Rational siRNA design for RNA interference," Nat Biotechnol, 22:326?30, 2004. 2. D.V. Morrissey et al., "Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus replication," Hepatology, 41:1349?56, June 2005. 3. J.M. Silva et al., "Second-generation shRNA libraries covering the mouse and human genomes," Nat Genet, 37:1281?8, November 2005. 4. R.A. Dickins et al., "Probing tumor phenotypes using stable and regulated synthetic microRNA precursors," Nat Genet, 37:1289?95, November 2005. 5. D. Dyxhoorn et al., "Killing the messenger: Short RNAs that silence gene expression," Nat Rev Mol Cell Biol, 4:457?67, 2003.
Table 1: Selected Online siRNA-Design Tools
Developer Name URL Ref.
Ambion siRNA Target Finder www.ambion.com/techlib/misc/siRNA_finder.html 1
Dharmacon
siDESIGN www.dharmacon.com/sidesign/ 2
GenScript SiRNA Construct Builder https://www.genscript.com/ssl-bin/app/rnai?op=known text
siRNA Target Finder https://www.genscript.com/ssl-bin/app/rnai 3
Invitrogen
Block-iT RNAi Designer https://rnaidesigner.invitrogen.com/rnaiexpress/ NA
Scionics Computer Innovation Deqor http://cluster-1.mpi-cbg.de/Deqor/deqor.html 4
Sigma Proligo
siRNA design tool www.proligo.com/pro_primprobes/PP_07-1_DS-siRNA.html 5
University of Tokyo siDirect http://design.rnai.jp/ 6
Wadsworth Center,
New York State Dept. Health

Sfold http://sfold.wadsworth.org/index.pl 7
Whitehead Institute siRNA Design http://jura.wi.mit.edu/bioc/siRNAext/ 8,9
Wistar Institute
siRNA Selector http://hydra1.wistar.upenn.edu/Projects/siRNA/ 10
References
2. A. Reynolds et al., "Rational siRNA design for RNA interference," Nat Biotechnol, 22:326-30, 2004.
3. L. Wang, F.Y. Mu, "A Web-based design center for vector-based siRNA and siRNA cassette," Bioinformatics, 20:1818-20, 2004.
4. A. Henschel et al., "DEQOR: A Web-based tool for the design and quality control of siRNAs," Nucleic Acids Res, 32:W113-20, 2004.
5. T. Tuschl et al., "The siRNA User Guide," http://www.rockefeller.edu/labheads/tuschl/sirna.html (revised May 6, 2004)
6. K. Ui-Tei et al., "Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference," Nucleic Acids Res, 32: 936-48, 2004.
7. Y. Ding, C.E. Lawrence, "Rational design of siRNAs with the Sfold software," in RNA Interference: From Basic Science to Drug Development, ed. Krishnarao Appasani, Cambridge University Press, 2005.
8. D.S. Schwarz et al., "Asymmetry in the assembly of the RNAi enzyme complex," Cell, 115:199-208, 2003.
9. A. Khovrova et al., "Functional siRNAs and miRNAs exhibit strand bias," Cell, 115:209-16, 2003.
10. N. Levenkova et al., "Gene specific siRNA selector," Bioinformatics, 20:430-2, 2004.
Table 2: siRNA Construction Kits
Company Kit siRNAs per kit (or yield in mg RNA/ml) Price (USD)
Ambion
www.ambion.com
MEGAscript RNAi Kit 20 $285
Silencer siRNA
Construction Kit
15 $535
Epicentre
www.epicentre.com
MessageMuter
ShRNAi Production Kit
10 $225
Genlantis
www.genlantis.com
Dicer siRNA
Generation Kit
5 $495
Turbo Dicer
siRNA Generation Kit
5 $550
Invitrogen
www.invitrogen.com
BLOCK-iT Complete
Dicer RNAi Kit
5 $746
BLOCK-iT Dicer
RNAi Transfection Kit
5 $510
BLOCK-iT RNAi
TOPO Transcription Kit
10 $304
New England Biolabs
www.neb.com
HiScribe RNAi
Transcription Kit
2 mg RNA $315
ShortCut RNAi Kit 100 RNA mg $420
Promega
www.promega.com
T7 RiboMAX
Express RNAi System
2-6 mg RNA $345
Roche Applied Science
www.roche-applied-science.com
X-treme Gene
siRNA Dicer Kit
10 $599
Spring Bioscience
www.springbio.com
Knock-Down
siRNA Kits (1-6)
5 $450
Stratagene
www.stratagene.com
RNAMaxx High
Yield Transcription Kit
80-100 mg/25 ml $201
Table 3: RNAi-Specific Viral Kits
Company Kit Size (reactions) Price (USD)
Ambion
www.ambion.com
pSilencer Adeno 1.0-CMV System 5 $520
pSilencer 5.1 Retro System 20 $250
Invitrogen
www.ambion.com
BLOCK-iT Adenoviral
RNAi Expression System
20 $941
BLOCK-iT Inducible
Lentviral RNAi System
20 $1594
BLOCK-iT Lentviral
RNAi Expression System
20 $1323
BLOCK-iT Lentviral
RNAi Gateway Vector Kit
20 $890
Imgenex
www.imgenex.com
pSuppressor Adeno-U6
RNAi Vector Kit
20 $465
pSuppressor Adeno RNAi Vector Kit 20 $465
pSuppressor Retro RNAi Vector Kit 20 $395
Welgen
www.welgeninc.com
pQuiet-U6 5 $650
Table 4: siRNA Transfection Reagents
Company text Maximum transfections/ml Price/ml (USD)
Ambion
www.ambion.com
siPORT neoFX
transfection reagent
1,250 $185
siPORT amine
transfection reagent
660 $345
siPORT lipid
transfection reagent
1,250 $210
B-Bridge International
www.b-bridge.com
Quick Step transfection reagent 2,000 $250
siFECTOR 1,000 $180
Bio-Rad Laboratories
www.bio-rad.com
siLentFect lipid transfection reagent 500 $250
Genlantis
www.genlantis.com
GeneSilencer siRNA transfection reagent 1,000 $387
Genospectra
www.genospectra.com
EXPRESS-si Delivery Kit 1,600 $700
Invitrogen
www.invitrogen.com
Lipofectamine 2000 667 $296
Mirus
www.mirusbio.com
TransIT-siQUEST 500 $318
TransIT-TKO 500 $318
New England Biolabs
www.neb.com
TransPass R1 200 $450
TransPass R2 400 $220
Novagen
www.novagen.com
Ribo-Juice siRNA Transfection Reagent 500 $335
Polypus Transfection
www.polypus-transfection.com
jetSI-ENDO 400 $233
Promega
www.promega.com
Codebreaker siRNA Transfection Reagent 250 $330
QIAGEN
www.qiagen.com
HiPerfect Transfection Reagent 166 $275
RNAiFect 170 $145
Roche Applied Science
www.roche-applied-science.com
X-treme Gene siRNA Transfection Reagent 400 $199
Stratagene
www.stratagene.com
GeneEraser siRNA Transfection Reagent (manufactured by Mirus) 500 $443
Upstate
www.upstate.com
siIMPORTER 267 $399
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