Gene Silencing by RNAi in Mammalian Cells

Frida Ponthan1, Narazah Mohd Yusoff2, Natalia Martinez Soria1, Olaf Heidenreich1, Kelly Coffey1

1 Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, 2 Advanced Medical & Dental Institute, Universiti Sains Malaysia, Pulau Pinang
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 26.2
DOI:  10.1002/0471142727.mb2602s111
Online Posting Date:  July, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


This unit provides information how to use short interfering RNA (siRNA) for sequence‐specific gene silencing in mammalian cells. Several methods for siRNA generation and optimization, as well as recommendations for cell transfection and transduction, are presented. © 2015 by John Wiley & Sons, Inc.

Keywords: RNA interference; siRNA; shRNA; transfection; electroporation; lentiviral transduction

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Liposome‐Mediated Forward and Reverse Transfection of Mammalian Cells with siRNA
  • Alternate Protocol 1: Electroporation of Mammalian Cells with siRNA
  • Alternate Protocol 2: Lentiviral Transduction of Suspension Cells using Spinfection
  • Support Protocol 1: Annealing Equimolar siRNA
  • Support Protocol 2: Production of Recombinant Pseudotyped Lentiviruses
  • Reagents and Solutions
  • Commentary
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Liposome‐Mediated Forward and Reverse Transfection of Mammalian Cells with siRNA

  • Mammalian cells to be transfected (e.g., LNCaP)
  • Complete medium for 293 cells (see recipe)
  • 20 μM annealed siRNA (see protocol 4)
  • Serum‐free RPMI medium
  • Lipofectamine RNAiMAX (Life Technologies)
  • 6‐well culture dishes
  • 1.5‐ml polypropylene tubes
  • Centrifuge
  • Additional reagents and equipment for mammalian cell tissue culture, including trypsinization ( appendix 3F; Phelan, ), northern blot hybridization (unit 4.9; Brown et al., ), RNase protection assay (unit 4.7; Gilman, ), RT‐PCR (unit 15.8; Bookout et al., ), and immunoblotting (unit 10.8; Gallagher et al., )
NOTE: Lipofectamine RNAiMAX is used in this protocol, as it works well for LNCaP cells. Other transfection reagents can also be used by following manufacturer's instructions. Lipofectamine RNAiMAX can be used to perform both forward and reverse transfections depending on user preference or requirements. For example, for large‐scale experiments, reverse transfection is preferred over forward transfection, as it is less labor intensive. Similarly, if the cell line is difficult to transfect, this method may improve the transfection efficiency.

Alternate Protocol 1: Electroporation of Mammalian Cells with siRNA

  Additional Materials (also see protocol 1Basic Protocol)
  • 4‐mm electroporation cuvettes (e.g., Peqlab 71‐2030)
  • Square‐wave electroporator (e.g., Fischer EPI 2500 ( or BioRad Gene Pulser Xcell)
  • Culture vessel
  • Additional reagents and equipment for electroporation (unit 9.3; Potter and Heller, )

Alternate Protocol 2: Lentiviral Transduction of Suspension Cells using Spinfection

  Additional Materials (also see protocol 1Basic Protocol)
  • 8 g/liter Polybrene
  • Phosphate‐buffered saline (PBS; appendix 22)
  • Virus particles containing shRNA expression vector
  • Appropriate selective antibiotic
  • Doxycycline
  • 24‐ and 48‐well culture plates
  • Centrifuge
  • Additional reagents and equipment for flow cytometry (Robinson et al., )

Support Protocol 1: Annealing Equimolar siRNA

  • siRNA sense and antisense strands (see Critical Parameters)
  • Annealing buffer: 100 mM NaCl in 25 mM HEPES, pH 7.5
  • Thermomixer Comfort (Eppendorf)
  • Additional reagents and equipment for nondenaturing polyacrylamide gel electrophoresis (unit 2.7; Chory and Pollard, ) and ethidium bromide staining (unit 4.9; Brown et al., )

Support Protocol 2: Production of Recombinant Pseudotyped Lentiviruses

  Additional Materials (also see protocol 1Basic Protocol)
  • 293T cells (ATCC #CRL‐3216 or DSMZ ACC‐635)
  • Complete medium for 293 cells (see recipe)
  • Envelope plasmid (e.g., pMD2.G; Addgene plasmid #12259)
  • Packaging plasmid (e.g., pCMVdeltaR8.91 or psPAX2; Addgene, plasmid #12260)
  • Second generation (with wild‐type 5′ LTR) lentiviral shRNA transfer vector
  • 2.5 mM HEPES‐buffered H 2O, pH 7.3 (store at 4°C)
  • 0.5 M CaCl 2 (store at 4°C)
  • 2× HeBS, pH 7.00 ( appendix 22)
  • 70% ethanol
  • 100‐mm tissue culture plates
  • Microscope
  • FACS tubes
  • 50‐ml conical centrifuge tubes (e.g., BD Falcon)
  • 0.45‐μm Acrodisc syringe filters (Pall Corporation)
  • Thick‐wall‐style (open‐top) polyallomer conical 30‐ml tubes (Beckman Instruments, cat. no. 358126)
  • Adapters (Beckman Instruments Inc. ref. 358156)
  • Beckman ultracentrifuge with swing‐out rotor (e.g., Beckman SW‐32 Ti)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Bakshi, R., Zaidi, S.K., Pande, S., Hassan, M.Q., Young, D.W., Montecino, M., Lian, J.B., van Wijnen, A.J., Stein, J.L., and Stein, G.S. 2008. The leukemogenic t(8;21) fusion protein AML1‐ETO controls rRNA genes and associates with nucleolar‐organizing regions at mitotic chromosomes. J. Cell Sci. 121:3981‐3990.
  Baulcombe, D. 2003. Overview of RNA interference and related processes. Curr. Protoc. Mol. Biol. 62:1.26.1‐
  Ben‐Ami, O., Friedman, D., Leshkowitz, D., Goldenberg, D., Orlovsky, K., Pencovich, N., Lotem, J., Tanay, A., and Groner, Y. 2013. Addiction of t(8;21) and inv(16) acute myeloid leukemia to native RUNX1. Cell Rep. 4:1131‐1143.
  Bookout, A.L., Cummins, C.L., Mangelsdorf, D.J., Pesola, J.M., and Kramer, M.F. 2006. High‐throughput real‐time quantitative reverse transcription PCR. Curr. Protoc. Mol. Biol. 73:15.8.1‐15.8.28.
  Brown, T., Mackey, K., and Du, T. 2004. Analysis of RNA by northern and slot blot hybridization. Curr. Protoc. Mol. Biol. 67:4.9.1‐4.9.19.
  Caplen, N.J., Parrish, S., Imani, F., Fire, A., and Morgan, R.A. 2001. Specific inhibition of gene expression by small double‐stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. U.S.A. 98:9742‐9747.
  Chiu, Y.L. and Rana, T.M. 2002. RNAi in human cells: Basic structural and functional features of small interfering RNA. Mol. Cell 10:549‐561.
  Chory, J. and Pollard, J.D. 1999. Separation of small DNA fragments by conventional gel electrophoresis. Curr. Protoc. Mol. Biol. 47:2.7.1‐2.7.8.
  Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. 2001. Duplexes of 21‐nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494‐498.
  Ellington, A. and Pollard, J.D. 1998. Purification of oligonucleotides using denaturing polyacrylamide gel electrophoresis. Curr. Protoc. Mol. Biol. 42:2.12.1‐2.12.7.
  Gallagher, S.R. 2011. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Curr. Protoc. Mol. Biol. 93:A.3D.1‐A.3D.14.
  Gallagher, S., Winston, S.E., Fuller, S.A., and Hurrell, J.G. 2008. Immunoblotting and immunodetection. Curr. Protoc. Mol. Biol. 83:10.8.1‐10.8.28.
  Gilman, M. 1993. Ribonuclease protection assay. Curr. Protoc. Mol. Biol. 24:4.7.1‐4.7.8.
  Gilman, M. 2002. Preparation of cytoplasmic RNA from tissue culture cells. Curr. Protoc. Mol. Biol. 58:4.1.1‐4.1.5.
  Hawley‐Nelson, P., Ciccarone, V., and Moore, M.L. 2008. Transfection of cultured eukaryotic cells using cationic lipid reagents. Curr. Protoc. Mol. Biol. 81:9.4.1‐9.4.17.
  Heidenreich, O., Benseler, F., Fahrenholz, A., and Eckstein, F. 1994. High activity and stability of hammerhead ribozymes containing 2′‐modified pyrimidine nucleosides and phosphorothioates. J. Biol. Chem. 269:2131‐2138.
  Heidenreich, O., Krauter, J., Riehle, H., Hadwiger, P., John, M., Heil, G., Vornlocher, H.P., and Nordheim, A. 2003. AML1/MTG8 oncogene suppression by small interfering RNAs supports myeloid differentiation of t(8;21)‐positive leukemic cells. Blood 101:3157‐3163.
  Holen, T., Amarzguioui, M., Wiiger, M.T., Babaie, E., and Prydz, H. 2002. Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. Nucleic Acids Res. 30:1757‐1766.
  Hornung, V., Guenthner‐Biller, M., Bourquin, C., Ablasser, A., Schlee, M., Uematsu, S., Noronha, A., Manoharan, M., Akira, S., de Fougerolles, A., Endres, S., and Hartmann, G. 2005. Sequence‐specific potent induction of IFN‐alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat. Med. 11:263‐270.
  Khvorova, A., Reynolds, A., and Jayasena, S.D. 2003. Functional siRNAs and miRNAs exhibit strand bias. Cell 115:209‐216.
  Kibbe, W.A. 2007. OligoCalc: An online oligonucleotide properties calculator. Nucleic Acids Res. 35:W43‐46.
  Krol, J., Loedige, I., and Filipowicz, W. 2010. The widespread regulation of microRNA biogenesis, function and decay. Nat. Rev. Genet. 11:597‐610.
  Lu, Z.J. and Mathews, D.H. 2008. OligoWalk: An online siRNA design tool utilizing hybridization thermodynamics. Nucleic Acids Res. 36:W104‐108.
  Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R., and Tuschl, T. 2002. Single‐stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110:563‐574.
  Novina, C.D., Murray, M.F., Dykxhoorn, D.M., Beresford, P.J., Riess, J., Lee, S.K., Collman, R.G., Lieberman, J., Shankar, P., and Sharp, P.A. 2002. siRNA‐directed inhibition of HIV‐1 infection. Nat. Med. 8:681‐686.
  Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
  Potter, H. and Heller, R. 2010. Transfection by electroporation. Curr. Protoc. Mol. Biol. 92:9.3.1‐9.3.10.
  Ptasinska, A., Assi, S.A., Mannari, D., James, S.R., Williamson, D., Dunne, J., Hoogenkamp, M., Wu, M., Care, M., McNeill, H., Cauchy, P., Cullen, M., Tooze, R.M., Tenen, D.G., Young, B.D., Cockerill, P.N., Westhead, D.R., Heidenreich, O., and Bonifer, C. 2012. Depletion of RUNX1/ETO in t(8;21) AML cells leads to genome‐wide changes in chromatin structure and transcription factor binding. Leukemia 26:1829‐1841.
  Rakheja, D., Chen, K.S., Liu, Y., Shukla, A.A., Schmid, V., Chang, T.C., Khokhar, S., Wickiser, J.E., Karandikar, N.J., Malter, J.S., Mendell, J.T., and Amatruda, J.F. 2014. Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis through distinct mechanisms in Wilms tumours. Nat. Commun. 2:4802.
  Robinson, J.P., Darzynkiewicz, Z., Nolan, J.P., Shankey, T.V., Telford, W., and Watkins, W. 2015. Current Protocols in Cytometry. John Wiley & Sons, New York.
  Saxena, S., Jonsson, Z.O., and Dutta, A. 2003. Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off‐target activity of small inhibitory RNA in mammalian cells. J. Biol. Chem. 278:44312‐44319.
  Scherr, M., Battmer, K., Ganser, A., and Eder, M. 2003. Modulation of gene expression by lentiviral‐mediated delivery of small interfering RNA. Cell Cycle 2:251‐257.
  Seizer, P., Schonberger, T., Schott, M., Lang, M.R., Langer, H.F., Bigalke, B., Kramer, B.F., Borst, O., Daub, K., Heidenreich, O., Schmidt, R., Lindemann, S., Herouy, Y., Gawaz, M., and May, A.E. 2010. EMMPRIN and its ligand cyclophilin A regulate MT1‐MMP, MMP‐9 and M‐CSF during foam cell formation. Atherosclerosis 209:51‐57.
  Thomas, M., Gessner, A., Vornlocher, H.P., Hadwiger, P., Greil, J., and Heidenreich, O. 2005. Targeting MLL‐AF4 with short interfering RNAs inhibits clonogenicity and engraftment of t(4;11)‐positive human leukemic cells. Blood 106:3559‐3566.
  Wilkinson, A.C., Ballabio, E., Geng, H., North, P., Tapia, M., Kerry, J., Biswas, D., Roeder, R.G., Allis, C.D., Melnick, A., de Bruijn, M.F., and Milne, T.A. 2013. RUNX1 is a key target in t(4;11) leukemias that contributes to gene activation through an AF4‐MLL complex interaction. Cell Rep. 3:116‐127.
  Wolfsberg, T.G. and Madden, T.L. 1999. Sequence similarity searching using the BLAST family of programs. Curr. Protoc. Mol. Biol. 46:19.3.1‐19.3.29.
  Yuan, B., Latek, R., Hossbach, M., Tuschl, T., and Lewitter, F. 2004. siRNA Selection Server: An automated siRNA oligonucleotide prediction server. Nucleic Acids Res. 32:W130‐134.
  Zeng, Y. and Cullen, B.R. 2002. RNA interference in human cells is restricted to the cytoplasm. RNA 8:855‐860.
Key References
  Elbashir, S.M., Harborth, J., Weber, K., and Tuschl, T. 2002. Analysis of gene function in somatic cells using small interfering RNAs. Methods 26:199‐213.
  The pioneers of mammalian RNA interference present a concise description of planning and performing siRNA experiments in this reference.
Internet Resources
  siRNA Selection Server of the Whitehead Institute.‐bin/server_exe/oligowalk/oligowalk_form.cgi
  TRC homepage.‐lentiviral‐shrna/
  GE Dharmacon inducible lentiviral shRNA expression.
PDF or HTML at Wiley Online Library