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Silencing of Gene Expression in Cultured Cells Using Small Interfering RNAs

Kumi Sakurai1,  Pritsana Chomchan1,  John J. Rossi1

1Beckman 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
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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

     
 
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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
     
 
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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 27.1.1 List of Web-Based siRNA Design Tools
    ToolsURLs
    siRNA Target Finderhttp://www.ambion.com/techlib/misc/siRNA_finder.html/
    BLOCK-IT RNAi Designerhttp://rnaidesigner.invitrogen.com/rnaiexpress/
    GeneAssist siRNA Workflow Builderhttp://www5.appliedbiosystems.com/tools/sirna/
    siDESIGN Centerhttp://www.dharmacon.com/DesignCenter/DesignCenterPage.aspx
    siRNA Target Designerhttp://www.promega.com/siRNADesigner/
    SpecificityServerhttp://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 (Basic 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% CO2 humidified incubator.


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

 Materials
  • siRNA-treated cells on 12-well plates (Basic 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 H2O (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, 2001), and real-time qPCR (Bookout et al., 2006)

Support Protocol 2: Assessing siRNA Transfection Efficiency by Immunoblotting

 Materials
  • siRNA-treated cell culture in 12-well plate (Basic 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 (Basic Protocol 1)
  • 10 µM irrelevant siRNA as negative control
  • psiCHECK-2 vector with target cloned into the MCS (psiYTC)
  • Stuffer DNA plasmid (see Basic 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% CO2 humidified incubator.


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

 Materials
  • Lyophilized single-stranded sense and antisense oligos (Basic Protocol 1)
  • RNase-free (e.g., DEPC-treated) H2O or TE buffer (appendix 2A)
  • 10× annealing buffer (see recipe)
  • Nuclease-free (e.g., DEPC-treated) H2O
  • 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 (Support Protocol 4)
  • 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.

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Figures

  • Figure 27.1.1
    Transfection of mammalian cells with siRNAs. Stepwise description of transfection of mammalian cells seeded onto a 12-well plate a day prior to transfection is shown above. After choosing five different concentrations of siRNAs to transfect the cells, mix siRNA and stuffer DNA in the Opti-MEM (or a serum-free medium) to bring the volume to 220 µl (step 1). Next, prepare the proper amount of Lipofectamine 2000 solution (two tubes of 720 µl for six tubes of siRNA/DNA mixes, step 2). After 5 min incubation, add 220 µl of Lipofectamine 2000 solution to each siRNA/DNA mix (step 3). Incubate for 20 min to let liposome formation, then add 200 µl of the liposome solution to transfect the cells (step 4). Transfection reagent preparations should be done in a tissue culture hood.

    View Image
  • Figure 27.1.2
    Detection of Cy3-labeled siRNA uptake by HEK293 cells. After 24 hr incubation, the transfection efficiency of 0, 1, 5, 25, 50, and 75 nM Cy3-labeled siRNAs were observed under a microscope with a UV lamp. Excitation wavelength of Cy3 is ~550 nm and emission wavelength is ~570 nm. Representative results are shown.

    View Image
  • Figure 27.1.3
    Effects of siRNA target gene down-regulation. (A) Determination of siRNA silencing efficiency by real-time qRT-PCR. Silencing of Gene A by si A or si B (10 nM) was determined with qRT-PCR. An irrelevant siRNA, si IR, was used as a negative control and used to normalize the Gene A expression level. The expression levels of the gene A was also analyzed relative to HRPT expression level set as 100%. The effects of siRNAs were determined in three independent transfections, and qRT-PCR reactions were performed in duplicate. (B) Determination of siRNA silencing efficiency by immunoblot analyses. Silencing of a gene A by si A or si B (10 nM) was determined at protein level. Quantification of protein expression levels are shown. An irrelevant siRNA, si IR, was used as a negative control and used to normalize the Gene A expression level. The expression levels of the Gene A was also analyzed relative to tubulin expression level set as 100%. The effects of siRNAs were determined in three independent transfections, and immunoblot analyses were performed independently.

    View Image
  • Figure 27.1.4
    A reporter plasmid psiCHECK-2 (Promega) containing a target site GFP S1. A reporter plasmid psiCHECK2-GFP-sense was prepared by cloning a PCR fragment that contains the target sequence complementary to siRNAs into the multiple cloning region of the psiCHECK-2 (S1 siRNA site). The psiCHECK-2 contains an additional internal control reporter gene, firefly luciferase (hluc+), which is used to normalize the Renilla luciferase activity. Depending on experimental settings, the siRNAs can be either cotransfected or transfected sequentially. Small interfering RNA silencing efficiency was evaluated by measuring the activities of firefly and Renilla luciferases sequentially from a single sample using a luminometer (http://www.promega.com).

    View Image
  • Figure 27.1.5
    Transfection of mammalian cells with psiCHECK2-GFP-sense and siRNAs (48-well plates). Stepwise procedures of transfection of mammalian cells seeded at two different cell densities (75% and 90%) a day prior to transfection are shown. After choosing five different concentrations of siRNAs (0 to 45 nM) to transfect the cells, prepare DNA master mixes and siRNA mixes separately in the Opti-MEM (or a serum-free medium) as indicated (steps 1 and 2). Next, add 70.4 µl of D1 to tubes containing 0 nM of siRNAs A, B, or C (tubes A1, B1, and C1). Repeat with D2 to D6 to prepare siRNA/DNA mixes (step 3). Prepare proper amount of Lipofectamine 2000 solution (three tubes of 576 µl for six tubes of siRNA/DNA mixes, step 4). After 5 min incubation, add 88 µl of Lipofectamine 2000 solution to each siRNA/DNA mix (step 5). Incubate for 20 min to allow liposome formation, then add 40 µl of the liposome solution to two wells on the 75% seeding density plate and two wells on the 90% seeding density plate (step 6). Transfection reagent preparations should be done in a tissue culture hood.

    View Image
  • Figure 27.1.6
    Determination of transfection efficiency by the dual-luciferase assays. The efficiency of siRNA silencing was evaluated by measuring the activities of firefly and Renilla luciferases. The relative luminescence units were normalized to the negative control (0 nM) set as 100%. Results showed concentration dependent silencing efficiency of the two siRNAs A and B. Once an optimal concentration of a siRNA is determined, perform your experiment with the highly potent siRNA(s) at optimal cell density and concentrations at least three times to assess reproducibility.

    View Image
  • Figure 27.1.7
    Verification of dsRNA integrity. The integrity of double-stranded RNAs was determined by gel electrophoresis. Either 1 pmol or 10 pmol of a 21-nt long dsRNA were loaded on a 8% non-denaturing polyacrylamide gel. Successful dsRNA annealing should result in a detectable higher-molecular-weight band and no remaining single-stranded oligo as a low-molecular-weight band, which was not detected in the gel.

    View Image

Literature Cited

Literature Cited
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    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.
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    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.
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    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.
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 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.

     
 
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