Differential Display of mRNA by PCR

Peng Liang1, Arthur B. Pardee2

1 Vanderbilt‐Ingram Cancer Center, Nashville, Tennessee, 2 Dana Farber Cancer Institute, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 25B.3
DOI:  10.1002/0471142727.mb25b03s56
Online Posting Date:  November, 2001
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Abstract

Formerly UNIT, this unit describes how differential display techniques allow the identification and subsequent isolation of differentially expressed genes that requires no knowledge of sequences, but rather PCR amplification using arbitrary oligonucleotides and high resolution polyacrylamide gel electrophoresis.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Total cellular human RNA (unit 4.2) or poly(A)+ RNA (unit 4.5)
  • 1 U/µl human placental RNase inhibitor
  • 10 U/µl DNase I (RNase‐free)
  • 0.1 M Tris⋅Cl, pH 8.3 ( appendix 22)
  • 0.5 M KCl
  • 15 mM MgCl 2
  • 3:1 (v/v) phenol/chloroform
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 100%, 70%, and 85% ethanol
  • Diethylpyrocarbonate (DEPC)–treated H 2O (unit 4.1)
  • 10 µM each degenerate anchored oligo(dT) primer set 5′‐T 12MN‐3′ (e.g., GenHunter): T 12MG, T 12MA, T 12MT, and T 12MC (M represents G, A, or C)
  • 5× MoMuLV reverse transcriptase buffer (unit 15.6)
  • 0.1 M dithiothreitol (DTT; appendix 22)
  • 250 µM and 25 µM 4dNTP mixes (unit 3.4)
  • 200 U/µl Moloney murine leukemia virus (MoMuLV) reverse transcriptase
  • 10× PCR amplification buffer (make as in unit 15.1, with 15 mM MgCl 2, but use only 0.1 mg/ml gelatin; store at −20°C)
  • 10 µCi/µl [α‐33P]dATP (>2000 Ci/mmol)
  • 2 µM arbitrary decamer (see Critical Parameters; e.g., GenHunter or Operon Technologies)
  • 5 U/µl Taq DNA polymerase
  • Mineral oil
  • recipeFormamide loading buffer (see recipe)
  • 10 mg/ml glycogen (DNA‐free)
  • 65°, 95°, 80°, and 100°C water baths
  • Thermal cycler
  • Whatman 3MM filter paper
  • Additional reagents and equipment for preparing total (unit 4.2) or poly(A)+ (unit 4.5) RNA, quantitating RNA ( appendix 3D), PCR (unit 15.1), agarose‐ formaldehyde gel electrophoresis (unit 4.9), denaturing PAGE (unit 7.6), autoradiography ( appendix 3A), agarose gel electrophoresis (unit 2.5), purifying DNA from agarose gels (unit 2.6), analysis of RNA by northern blot analysis (unit 4.9), screening libraries using oligonucleotide probes (unit 6.3), cloning PCR products (unit 15.4), and dideoxy DNA sequencing (unit 7.4)
CAUTION: This procedure should be performed only by personnel trained in the proper use of 33P isotope and in NRC licensed sites. Standard precautions to prevent excessive exposure and radioactive contamination of personnel and equipment should be followed at all times.NOTE: Experiments involving RNA require careful technique to prevent RNA degradation (unit 4.1).
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Figures

Videos

Literature Cited

   Comes, A., Humbert, J., and Laurent, F. 1997. Rapid cloning of PCR‐derived RAPD probes. BioTechniques 23:210‐212.
   Hedrick, S.M., Cohen, D.I., Nielsen, E.A., and Davis, M.M. 1984. Isolation of cDNA clones encoding T cell specific membrane associated proteins. Nature 308:149‐153.
   Lee, S.W., Tomasetto, C., and Sager, R. 1991. Positive selection of candidate tumor‐suppressor genes by subtractive hybridization. Proc. Natl.Acad. Sci. U.S.A. 88:2825‐2829.
   Liang, P. and Pardee, A.B. 1993. Distribution and cloning of eukaryotic mRNAs by means of differential display: Refinements and optimization. Nucl. Acids Res. 21:3269‐3275.
   Liang, P., Averboukh, L., and Pardee, A.B. 1993. Distribution and cloning of eukaryotic mRNAs by means of differential display: Refinements and optimization. Nucl. Acids Res. 21:3269‐3275.
   Liang, P., Zhu, W., Zhang, X., Gui, Z., O'Connell, R.P., Averboukh, L., Want, F., and Pardee, A.B. 1994. Differential display using one‐base anchored oligo‐dT primers. Nucleic Acids Res. 22:5763‐5764.
   Lisitsyn, N.A. 1995. Representational difference analysis: Finding the difference between genomes. Trends Genet. 11:303‐307.
   Martin, K.J., Kwan, C.‐P., O'Hare, M.J., Pardee, A.B., and Sager, R. 1998. Identification and verification of differential display cDNAs using gene‐specific primers and hybridization arrays. BioTechniques 24:1018‐1026.
   Miele, G., MacRae, L., McBride, D., Manson, J., and Clinton, M. 1998. Elimination of flase positives generated through PCR reamplification of differential display cDNA. BioTechniques 25:138‐144.
   Pesole, G., Liuni, S., Grillo, G., Belichared, P., Trenkle, T., Walse, J., and McClelland, M. 1998. GeneUp: A program to select short PCR primer pairs that occur in multiple members of sequence lists. BioTechniques 25:112‐123.
   Prasher, Y. and Weissman, S.M. 1996. Analysis of differential gene expression by display of 3′ end restriction fragments of cDNAs. Proc. Natl. Acad. Sci. U.S.A. 93:659‐663.
   Sager, R. 1997. Expression genetics: Shifting the focus from DNA to RNA. Proc. Natl. Acad. Sci. U.S.A. 94:952‐955.
   Steeg, P.S., Bevilacqua, G., Kopper, L., Thorgeirson, U.P., Talmadge, J.E., Liotta, L.A., and Sobel, M.E. 1988. Evidence for a novel gene associated with low tumor metastatic potential. J. Natl. Cancer Inst. 80:200‐204.
   Trentmann, S.M., van der Dnapp, E., and Kende, H. 1995. Alternatives to 35S as a label for the differential display of eukaryotic messenger RNA, Science 267:1186‐1187.
   Wang, W. and Feurstein, G.Z. 1995. Direct sequencing of DNA isolated from mRNA differential display. BioTechniques 18:448‐453.
   Wodicka, L., Dong, H., Mittmann, M., Ho, M‐H., and Lockhart, D.J. 1997. Genome‐wide expression monitoring in Saccharomyces cerevisiae. Nature Biotechnology 15:1359‐1367.
   Wybranietz, W. and Lauer, U. 1998. Distincet combination of purification methods dramatically improves cohesive‐end subcloning of PCR products. BioTechniques 24:578‐580.
   Zhang, H., Zhang, R., and Liang, P. 1996. Differential screening of gene expression difference enriched by differential display. Nucleic Acids Res. 24:2454‐2455.
   Zhang, L., Zhou, V.E., Velculescu, S.E., Kern, R.H., Hruban, S.R., Hamilton, B., Volgelstein, B., and Kinzler, K.W. 1997. Gene expression profiles in normal and cancer cells. Science 276:1268‐1272.
   Zhao, S., Ooi, S.L., and Pardee, A.B. 1995. New primer strategy improves precision of differential display. BioTechniques 18:842‐850.
   Zhao, S., Ooi, S.L., and Pardee, A.B. 1996. Three methods for the identification of true positive cloned cDNA fragments in differential display. BioTechniques 20:400‐402.
Key Reference
   Liang et al., 1993. See above.
  Uses the protocol outlined here and presents examples of data generated.
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