Enzymatic Amplification of RNA by PCR (RT‐PCR)

Stephen M. Beverly1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 15.5
DOI:  10.1002/0471142727.mb1505s56
Online Posting Date:  November, 2001
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Abstract

Methods for enzymatic amplification of RNA by the polymerase chain reaction (RT‐PCR) are highlighted in this unit. The is especially useful for rare RNAs because all steps (annealing, reverse transcription, and amplification) are performed under optimal conditions, thereby maximizing efficiency and recovery. These are also important considerations when amplifying heterogeneous RNA populations or large RNAs. Although there are many commercially available kits for this procedure, the protocols listed here are good solid protocols that will work and that have withstood the test of time.

     
 
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Table of Contents

  • Basic Protocol 1: PCR Amplification of RNA Under Optimal Conditions
  • Alternate Protocol 1: Avoiding Lengthy Coprecipitation and Annealing Steps
  • Alternate Protocol 2: Introducing cDNA Directly into the Amplification Step
  • Support Protocol 1: Rapid Preparation of Crude RNA
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: PCR Amplification of RNA Under Optimal Conditions

  Materials
  • Poly(A)+ (unit 4.5) or crude RNA (see protocol 4)
  • 25 µg/ml cDNA primer in H 2O
  • 3 M sodium acetate, pH 5.5 ( appendix 22)
  • 100% and 70% ethanol
  • 400 mM Tris⋅Cl, pH 8.3 ( appendix 22)
  • 400 mM KCl ( appendix 22)
  • recipeReverse transcriptase buffer (see recipe)
  • 32 U/µl AMV reverse transcriptase (unit 3.7; Boehringer‐Mannheim)
  • 10 mM Tris⋅Cl/10 mM EDTA, pH 7.5
  • Phenol buffered with 10 mM Tris⋅Cl/10 mM EDTA, pH 7.5 (store at room temperature)
  • 24:1 chloroform/isoamyl alcohol (unit 2.1)
  • ∼150 µg/ml amplification primers in H 2O (∼20 µM each)
  • 5 mM 4dNTP mix (5 mM each dNTP in H 2O; unit 3.4)
  • 10× PCR amplification buffer (unit 15.1)
  • Taq DNA polymerase (unit 15.1)
  • Mineral oil
  • Additional reagents and equipment for PCR, (unit 15.1), preparation of poly (A)+ RNA (unit 4.5), total RNA (units 4.2 4.4), or cytoplasmic RNA (unit 4.1), and for agarose or nondenaturing polyacrylamide gel electrophoresis (units 2.5 & 2.7)
NOTE: Reagents and solutions used for RNA procedures should be prepared using standard methods for handling RNA (unit 4.1).

Alternate Protocol 1: Avoiding Lengthy Coprecipitation and Annealing Steps

  Materials
  • Phosphate‐buffered saline (PBS; appendix 22)
  • recipeDiethylpyrocarbonate (DEPC) solution (see recipe)
CAUTION: DEPC is volatile and toxic and should be handled with precautions. DEPC is also rapidly inactivated in aqueous buffers and in the presence of Tris⋅Cl, so work quickly.
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Figures

Videos

Literature Cited

Literature Cited
   Berchtold, M.W. 1989. A simple method for direct cloning and sequencing cDNA by the use of a single specific oligonucleotide and oligo(dT) in a polymerase chain reaction (PCR). Nucl. Acids Res. 17:453.
   Frohman, M.A., Dush, M.K., and Martin, G.R. 1988. Rapid production of full length cDNAs from rare transcripts: Amplification using a single gene‐specific oligonucleotide primer. Proc. Natl. Acad. Sci. U.S.A. 85:8998‐9002.
   Higuchi, R. 1989. Simple and rapid preparation of samples for PCR. In PCR Technology: Principles and Applications for DNA Amplification (H.A. Ehrlich, ed.) pp. 31‐38. Stockton Press, New York.
   Kapler, G.M., Coburn, C.M., and Beverley, S.M. 1990. Transfection of the human parasite Leishmania delineates a 30 kb region sufficient for extra‐chromosomal replication and expression. Mol. Cell. Biol. 10:1084‐1094.
Key Reference
   Frohman et al., 1988. See above.
  Outlines several strategies for using PCR to analyze RNA structure and for production of full‐length cDNAs.
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