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
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Abstract

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

     
 
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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
     
 
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Materials

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

  Materials
  • 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 (http://www.electroporation.eu) 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

  Materials
  • 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)
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Figures

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Literature Cited

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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
  http://sirna.wi.mit.edu/reference.php
  siRNA Selection Server of the Whitehead Institute.
  http://rna.urmc.rochester.edu/cgi‐bin/server_exe/oligowalk/oligowalk_form.cgi
  OligoWalk.
  http://www.broadinstitute.org/rnai/public/
  TRC homepage.
  http://dharmacon.gelifesciences.com/shrna/tripz‐lentiviral‐shrna/
  GE Dharmacon inducible lentiviral shRNA expression.
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