Differential Display of mRNA by PCR

F.J. Livesey1, C.P. Hunt2

1 Trinity College, Dublin, 2 Medical Research Council Laboratory of Molecular Biology, Cambridge
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.2
DOI:  10.1002/0471142301.ns0502s00
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 approach described here is a modification of the original method, referred to as single base‐anchored differential display. The basic protocol describes the actual differential display PCR reaction along with details of the identification, reamplification, and cloning of candidate differentially expressed genes. The 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
  • 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 Background Information)
  • 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 2A)
  • 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 2A)
  • 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, gel drying, agarose gel electrophoresis, and recovery of DNA from agarose (see CPMB UNITS , , & and appendix 1A in this manual)
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 up in DEPC‐treated water (see appendix 2A 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 ( appendix 2A)
  • 0.1 M Tris⋅Cl, pH 8.3 ( appendix 2A)
  • 0.5 M KCl
  • 15 mM MgCl 2
  • Nuclease‐free water (e.g., water treated with DEPC; appendix 2A)
  • Total cellular RNA from the tissues to be studied (see CPMB UNIT and appendix 1A in this manual)
  • 3:1 (v/v) buffered phenol ( appendix 2A)/chloroform
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 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
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Figures

Videos

Literature Cited

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
   Liang et al., 1994. See above.
  Describes the second, more reproducible version of differential display.
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