Differential Display of mRNA by PCR

F.J. Livesey1, S.P. Hunt2

1 Harvard Medical School, Boston, Massachusetts, 2 University College London, London, United Kingdom
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 11.5
DOI:  10.1002/0471142905.hg1105s10
Online Posting Date:  May, 2001
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Abstract

This unit outlines the polymerase chain reaction (PCR)‐based technique of mRNA differential display, which identifies genes that are differentially expressed between cells or tissues. The basic protocol describes the actual differential display PCR reaction along with details of the identification, reamplification, and cloning of candidate differentially expressed genes. A support protocol provides instructions on removing contaminating genomic DNA from the RNA samples and reverse transcribing the purified RNA to produce the cDNA used in the subsequent PCR reactions.

     
 
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Table of Contents

  • Basic Protocol 1: Differential Display PCR
  • Support Protocol 1: Preparation of cDNA for Differential Display PCR
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Differential Display PCR

  Materials
  • cDNA (see protocol 2)
  • 2 µM single base–anchored oligo(dT) primers (Hind‐dT 11M):
  • Hind‐dT 11A: AAG CTT TTT TTT TTT A
  • Hind‐dT 11C: AAG CTT TTT TTT TTT C
  • Hind‐dT 11G: AAG CTT TTT TTT TTT G
  • 2 µM arbitrary 13‐base primers (see )
  • 5 U/µl AmpliTaq DNA polymerase (Perkin‐Elmer) and 10× buffer
  • 10 µCi/µl [α‐33P]dATP (1000 to 3000 Ci/mmol; DuPont NEN)
  • 25 and 250 µM 4dNTP mixtures
  • 25 mM MgCl 2
  • Nuclease‐free water (e.g., water treated with DEPC; appendix 2D)
  • Mineral oil
  • Denaturing gel loading buffer (see recipe)
  • 1 µg/µl molecular biology–grade glycogen (Boehringer Mannheim)
  • 3 M sodium acetate, pH 5.2 ( appendix 2D)
  • 100% ethanol
  • 80% ethanol made with nuclease‐free water, ice cold
  • Cloning vector (for cloning PCR product)
  • Thermal cycler
  • 80° and 100°C heating blocks or water baths
  • X‐ray film (BioMax, Kodak)
  • Additional reagents and equipment for polyacrylamide/urea gel electrophoresis ( appendix 3F or CPMB UNIT ), agarose gel electrophoresis (unit 2.7), and recovery of DNA from agarose (see unit 5.4 or CPMB UNIT )
NOTE: All plastics should be autoclaved to destroy contaminating nucleases. All aqueous solutions should be made nuclease free by treating with 1 ml diethylpyrocarbonate (DEPC) per liter of solution and autoclaving, or by making solutions in DEPC‐treated water (see appendix 2D for details of DEPC treatment).CAUTION: DEPC is a potent carcinogen and should be handled with care.CAUTION: Do not use [α‐35S]dATP as the radionucleotide to label the PCR products, as it can degrade at high temperatures, liberating the 35S. Use [α‐33P]dATP instead.

Support Protocol 1: Preparation of cDNA for Differential Display PCR

  Materials
  • RNasin recombinant RNase inhibitor (Promega)
  • DNase I, RNase‐free (see recipe)
  • 0.1 M Tris⋅Cl, pH 8.3 ( appendix 2D)
  • 0.5 M KCl
  • 15 mM MgCl 2
  • Nuclease‐free water (e.g., water treated with DEPC; appendix 2D)
  • Total cellular RNA from the tissues to be studied (see e.g., unit 10.4 or CPMB UNIT )
  • 3:1 (v/v) buffered phenol ( appendix 3C)/chloroform
  • 3 M sodium acetate, pH 5.2 ( appendix 2D)
  • 100% ethanol
  • 80% ethanol made with nuclease‐free water, ice cold
  • 2 µM single base–anchored oligo(dT) primers (Hind‐dT 11M):
  • Hind‐dT 11A: AAG CTT TTT TTT TTT A
  • Hind‐dT 11C: AAG CTT TTT TTT TTT C
  • Hind‐dT 11G: AAG CTT TTT TTT TTT G
  • 200 U/µl Moloney murine leukemia virus (MMLV) reverse transcriptase (SuperScript II, Life Technologies) and 5× buffer
  • 0.1 M dithiothreitol (DTT)
  • 250 µM 4dNTP mixture
  • 37°, 70°, and 95°C heating blocks or water baths
  • Additional reagents and equipment for spectrophotometric quantition of RNA ( appendix 3D)
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Figures

Videos

Literature Cited

   Bauer, D., Muler, H., Reich, J., Riedel, H., Ahrenkiel, V., Warthoe, P., and Strauss, M. 1993. Identification of differentially expressed mRNA species by an improved differential display technique (DDRT‐PCR). Nucl. Acids Res. 21:4272‐4280.
   Liang, P. and Pardee, A.B. 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967‐971.
   Liang, P., Zhu, W., Zhang, X., Guo, Z., O'Connell, R.P., Averboukh, L., Wang, F., and Pardee, A.B. 1994. Differential display using one‐base anchored oligo‐dT primers. Nucl. Acids Res. 22:5763‐5764.
Key Reference
   Cho, Y.‐J. and Liang, P. 2000. Differential display analysis of alteration in gene expression. In Functional Genomics, A Practical Approach (S.P. Hunt and F.J. Livesey, eds). Oxford University Press, UK.
  Recent discussion of the uses of the method by the developers of the technique.
   Liang et al., 1994 See above.
  Describes the second, more reproducible version of differential display.
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