Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs

Kumi Sakurai1, Pritsana Chomchan1, John J. Rossi1

1 Beckman Research Institute of City of Hope, Duarte, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 27.1
DOI:  10.1002/0471143030.cb2701s47
Online Posting Date:  June, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The discovery of RNA interference (RNAi) and related small RNA–mediated regulatory pathways has significantly altered the understanding of gene regulation in eukaryotic cells. In the RNAi pathway, small interfering RNAs (siRNAs) ∼21 to 23 nucleotides in length serve as the regulatory molecules that guide and induce sequence‐specific gene silencing. The use of siRNA‐mediated silencing as a tool for investigating gene function is well established in cultured mammalian cells. This unit provides basic approaches to explore the field of RNAi, and hopes to address the importance of optimizing transfection conditions after empirical determinations in order to understand various degrees of silencing efficiency. Curr. Protoc. Cell Biol. 47:27.1.1‐27.1.28. © 2010 by John Wiley & Sons, Inc.

Keywords: RNAi; small silencing RNAs; gene silencing; transfection; mammalian cell culture

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Design of 21‐Nucleotide‐Long siRNA (21‐MER)
  • Basic Protocol 2: Determining Optimal Transfection Conditions
  • Support Protocol 1: Assessing siRNA Transfection Efficiency by RT‐PCR
  • Support Protocol 2: Assessing siRNA Transfection Efficiency by Immunoblotting
  • Support Protocol 3: Assessing siRNA Transfection Efficiency by a Dual‐Reporter Assay System
  • Support Protocol 4: Annealing Single‐Stranded Oligos for a Double‐Stranded RNA
  • Support Protocol 5: Checking the Integrity of dsRNAs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Design of 21‐Nucleotide‐Long siRNA (21‐MER)

  Materials
  • Gene of interest
  • Web‐based siRNA design program (e.g., Table 27.1.1)
  • Web‐based specificity program (e.g., SpecificityServer; Table 27.1.1)
    Table 7.1.1   MaterialsList of Web‐Based siRNA Design Tools

    Tools URLs
    siRNA Target Finder http://www.ambion.com/techlib/misc/siRNA_finder.html/
    BLOCK‐IT RNAi Designer http://rnaidesigner.invitrogen.com/rnaiexpress/
    GeneAssist siRNA Workflow Builder http://www5.appliedbiosystems.com/tools/sirna/
    siDESIGN Center http://www.dharmacon.com/DesignCenter/DesignCenterPage.aspx
    siRNA Target Designer http://www.promega.com/siRNADesigner/
    SpecificityServer http://informatics‐eskitis.griffith.edu.au/SpecificityServer/

Basic Protocol 2: Determining Optimal Transfection Conditions

  Materials
  • Mammalian cell line cultured in the appropriate growth medium in 10‐cm2 dishes
  • Calcium‐ and magnesium‐free Dulbecco's phosphate‐buffered saline (CMF‐DPBS; Cellgro, cat. no. 21‐031 CV, or see appendix 2A), 37°C
  • 1× trypsin/EDTA solution (e.g., Invitrogen; also see unit 1.1), 37°C
  • Complete DMEM with 10% (v/v) FBS (see recipe), 37°C
  • Complete DMEM with 10% (v/v) FBS (see recipe), without antibiotics, 37°C
  • Trypan blue staining solution (unit 1.1)
  • 10 µM siRNA working solution ( protocol 1)
  • 10 µM irrelevant siRNA as a negative control
  • 10 µM fluorescent dye (e.g., Cy3 or FITC)–labeled siRNA as a transfection control
  • 250 ng/µl stuffer DNA plasmid (e.g., pBluescript, pCR2.1; Clontech, Invitrogen)
  • Opti‐MEM I (a reduced‐serum medium from Invitrogen), or serum‐free growth medium
  • Lipofectamine 2000 (Invitrogen)
  • Microscope
  • Hemacytometer with coverslip (Figure 1.1.1)
  • 12‐well tissue culture plates
  • Standard microscope and UV lamp or fluorescence microscope
  • Additional reagents and equipment for basic cell culture techniques including trypsinization and counting viable cells on a hemacytometer by trypan blue exclusion (unit 1.1)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All cell culture incubations should be carried out in a 37°C, 5% CO 2 humidified incubator.

Support Protocol 1: Assessing siRNA Transfection Efficiency by RT‐PCR

  Materials
  • siRNA‐treated cells on 12‐well plates ( protocol 2)
  • RNA STAT‐60 (Tel‐Test, Inc., http://www.tel‐test.com/) at 4°C
  • Chloroform
  • 75% ethanol made with nuclease‐free or diethylpyrocarbonate (DEPC)‐treated H 2O (see appendix 2A fro DEPC treatment)
  • Nuclease‐free or DEPC‐treated water ( appendix 2A)
  • 2 U/µl DNase I (RNase‐free) and 10× DNase buffer (Ambion)
  • 1 U/µl RNase inhibitor (RNasin, Promega)
  • Random hexamer primers [or oligo(dT) 12‐18]
  • 10 mM dNTP mix(10 mM dATP, dCTP, dGTP, and dTTP)
  • 5× first‐strand buffer (Invitrogen)
  • 0.1 M DTT
  • MMLV reverse transcriptase (Invitrogen)
  • Forward and reverse PCR primers
  • iQ SYBR Green Supermix (for real time qPCR; BioRad)
  • Refrigerated centrifuge
  • NanoDrop 1000 (Thermo Fisher Scientific) or UV/Vis spectrophotometer
  • PCR tubes
  • 65° and 80°C water baths or heating block
  • Additional reagents and equipment for spectrophotometric determination of RNA concentration ( appendix 3D), standard PCR (Kramer and Coen, ), and real‐time qPCR (Bookout et al., )

Support Protocol 2: Assessing siRNA Transfection Efficiency by Immunoblotting

  Materials
  • siRNA‐treated cell culture in 12‐well plate ( protocol 2)
  • Mock‐treated cell culture
  • Calcium‐ and magnesium‐free Dulbecco's phosphate‐buffered saline (CMF‐DPBS; Cellgro, cat. no. 21‐031 CV, or see appendix 2A), 4°C
  • RIPA buffer (see recipe) with freshly added 1× protease inhibitor cocktail (Roche), ice cold
  • End‐over‐end rotator (e.g., Labquake from Thermo Scientific)
  • Additional reagents and equipment for Bradford protein assay ( appendix 3H) and immunoblotting (unit 6.2)

Support Protocol 3: Assessing siRNA Transfection Efficiency by a Dual‐Reporter Assay System

  Materials
  • 10 µM siRNA working solution ( protocol 1)
  • 10 µM irrelevant siRNA as negative control
  • psiCHECK‐2 vector with target cloned into the MCS (psiYTC)
  • Stuffer DNA plasmid (see protocol 2)
  • Opti‐MEM I (a reduced‐serum medium from Invitrogen), or serum‐free growth medium
  • Lipofectamine 2000 (Invitrogen)
  • Mammalian cells seeded in two 48‐well plates
  • Complete medium with 10% FBS (e.g., complete DMEM/10% FBS; see recipe), without antibiotics, 37°C
  • Dual‐Luciferase Reporter Assay System (Promega)
  • Orbital shaker
  • Luminometer (e.g., Veritas Microplate Luminometer)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.NOTE: All cell culture incubations should be carried out in a 37°C, 5% CO 2 humidified incubator.

Support Protocol 4: Annealing Single‐Stranded Oligos for a Double‐Stranded RNA

  Materials
  • Lyophilized single‐stranded sense and antisense oligos ( protocol 1)
  • RNase‐free (e.g., DEPC‐treated) H 2O or TE buffer ( appendix 2A)
  • 10× annealing buffer (see recipe)
  • Nuclease‐free (e.g., DEPC‐treated) H 2O
  • 95°C heat block
  • NanoDrop 1000 (Thermo Fisher Scientific) or UV/Vis spectrophotometer
  • Additional reagents and equipment for spectrophotometric determination of RNA concentration ( appendix 3D)

Support Protocol 5: Checking the Integrity of dsRNAs

  Materials
  • 10× TBE buffer ( appendix 2A)
  • 40% (w/v) 19:1 acrylamide:bisacrylamide (AC:BC) solution
  • TEMED
  • 10% (w/v) ammonium persulfate (APS) solution
  • Annealed dsRNAs ( protocol 6)
  • 4× native gel loading dye (see recipe)
  • 10 mg/ml ethidium bromide solution
  • 14 × 16–cm gel electrophoresis apparatus
  • Power supply
  • Flat gel loading tips
  • UV lamp
NOTE: When a minigel is used, adjust the volume and time of gel running.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Amarzguioui, M., Holen, T., Babaie, E., and Prydz, H. 2003. Tolerance for mutations and chemical modifications in a siRNA. Nucleic Acids Res. 31:589‐595.
   Bernstein, E., Caudy, A.A., Hammond, S.M., and Hannon, G.J. 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363‐366.
   Bookout, A.L., Cummins, C.L., Kramer, M.F. Pesola, J.M., and Mangelsdorf, D.J. 2006. High‐throughput real‐time quantitative reverse transcription PCR. Curr. Protoc. Mol. Biol. 73:15.8.1‐15.8.28.
   Chalk, A.M. and Sonnhammer, E.L. 2008. siRNA specificity searching incorporating mismatch tolerance data. Bioinformatics 24:1316‐1317.
   Chalk, A.M., Warfinge, R.E., Georgii‐Hemming, P., and Sonnhammer, E.L. 2005. siRNAdb: A database of siRNA sequences. Nucleic Acids Res. 33:D131‐134.
   Ding, Y., Chan, C.Y., and Lawrence, C.E. 2004. Sfold web server for statistical folding and rational design of nucleic acids. Nucleic Acids Res. 32:W135‐141.
   Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. 2001a. Duplexes of 21‐nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494‐498.
   Elbashir, S.M., Lendeckel, W., and Tuschl, T. 2001b. RNA interference is mediated by 21‐ and 22‐nucleotide RNAs. Genes Dev. 15:188‐200.
   Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendeckel, W., and Tuschl, T. 2001c. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20:6877‐6888.
   Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. 1998. Potent and specific genetic interference by double‐stranded RNA in Caenorhabditis elegans. Nature 391:806‐811.
   Gong, W., Ren, Y., Zhou, H., Wang, Y., Kang, S., and Li, T. 2008. siDRM: An effective and generally applicable online siRNA design tool. Bioinformatics 24:2405‐2406.
   Gregory, R.I., Chendrimada, T.P., Cooch, N., and Shiekhattar, R. 2005. Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123:631‐640.
   Heale, B.S., Soifer, H.S., Bowers, C., and Rossi, J.J. 2005. siRNA target site secondary structure predictions using local stable substructures. Nucleic Acids Res. 33:e30.
   Kramer, M.F. and Coen, D.M. 2001. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol. 15.1.1‐15.1.14.
   Liu, J., Carmell, M.A., Rivas, F.V., Marsden, C.G., Thomson, J.M., Song, J.J., Hammond, S.M., Joshua‐Tor, L., and Hannon, G.J. 2004. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305:1437‐1441.
   Liu, Q., Rand, T.A., Kalidas, S., Du, F., Kim, H.E., Smith, D.P., and Wang, X. 2003. R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science 301:1921‐1925.
   Meister, G., Landthaler, M., Patkaniowska, A., Dorsett, Y., Teng, G., and Tuschl, T. 2004. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Molec. Cell 15:185‐197.
   Owczarzy, R., Tataurov, A.V., Wu, Y., Manthey, J.A., McQuisten, K.A., Almabrazi, H.G., Pedersen, K.F., Lin, Y., Garretson, J., McEntaggart, N.O., Sailor, C.A., Dawson, R.B., and Peek, A.S. 2008. IDT SciTools: A suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res. 36:W163‐W169.
   Ren, Y., Gong, W., Zhou, H., Wang, Y., Xiao, F., and Li, T. 2009. siRecords: A database of mammalian RNAi experiments and efficacies. Nucleic Acids Res. 37:D146‐D149.
   Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W.S., and Khvorova, A. 2004. Rational siRNA design for RNA interference. Nat. Biotechnol. 22:326‐330.
   Rivas, F.V., Tolia, N.H., Song, J.J., Aragon, J.P., Liu, J., Hannon, G.J., and Joshua‐Tor, L. 2005. Purified Argonaute2 and an siRNA form recombinant human RISC. Nat. Struct. Molec. Biol. 12:340‐349.
   Rose, S.D., Kim, D.H., Amarzguioui, M., Heidel, J.D., Collingwood, M.A., Davis, M.E., Rossi, J.J., and Behlke, M.A. 2005. Functional polarity is introduced by Dicer processing of short substrate RNAs. Nucleic Acids Res. 33:4140‐4156.
   Shah, J.K., Garner, H.R., White, M.A., Shames, D.S., and Minna, J.D. 2007. sIR: siRNA Information Resource, a web‐based tool for siRNA sequence design and analysis and an open access siRNA database. BMC Bioinformatics 8:178.
   Siolas, D., Lerner, C., Burchard, J., Ge, W., Linsley, P.S., Paddison, P.J., Hannon, G.J., and Cleary, M.A. 2005. Synthetic shRNAs as potent RNAi triggers. Nat. Biotechnol. 23:227‐231.
   Ui‐Tei, K., Naito, Y., Takahashi, F., Haraguchi, T., Ohki‐Hamazaki, H., Juni, A., Ueda, R., and Saigo, K. 2004. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res. 32:936‐948.
   Yiu, S.M., Wong, P.W., Lam, T.W., Mui, Y.C., Kung, H.F., Lin, M., and Cheung, Y.T. 2005. Filtering of ineffective siRNAs and improved siRNA design tool. Bioinformatics 21:144‐151.
   Zamore, P.D., Tuschl, T., Sharp, P.A., and Bartel, D.P. 2000. RNAi: Double‐stranded RNA directs the ATP‐dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25‐33.
Internet Resources
  http://genome.ucsc.edu/cgi‐bin/hgGateway
  Source for mRNA sequences of genes.
  http://www.idtdna.com/Scitools/Applications/RNAi/RNAi.aspx
  Site for assistance with RNAi design.
  http://www1.infosci.coh.org/hpcdispatcher/
  Home of RNAi Site selector which considers secondary structure of target transcripts.
  http://www.invitrogen.com
  Cell‐type specific transfection protocols for stealth RNAi and siRNA.
  http://www.promega.com/paguide/chap8.htm
  Protocols and applications guide.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library