Advertisement

Nonradioactive Differential Display of Messenger RNA

  • Thomas C. G. Bosch
  • Jan U. Lohmann
Protocol
  • 81 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Changes in cell behavior are driven by changes in gene expression. Thus, in order to understand the mechanisms regulating cell behavior, one has to identify and characterize differentially expressed genes. Standard methods currently used to isolate differentially expressed genes include subtractive hybridization (1,2), differential hybridization (3), and single-cell polymerase chain reaction (PCR) (4). Differential display of mRNA by PCR (DD-PCR) is a new and powerful procedure for quantitative detection of differentially expressed genes (5,6). Advantages over alternative approaches include: quantitative identification of differences in gene expression between different cell fractions; simultaneous detection of both upregulation and downregulation of genes; requirement of only small amounts of messenger RNA; and drastically reduced time of analysis. Since on average there are about 15,000 individual mRNA species present in any individual cell, some steps of selection have to be taken before the transcript population can be displayed simultaneously. Therefore, in the DD-PCR procedure mRNAs from different cell fractions are reverse transcribed (RT) using an oligo-dT-NN anchor primer. Since this 3′ primer will hybridize only with transcripts carrying the corresponding two NN-bases in front of the poly(A) tail, only 1/12 (i.e., about 1200) of all transcripts are reverse transcribed into cDNA. Accordingly, for reverse transcription of all messages 12 different 3′ primer have to be used independently. PCR of the resulting cDNA is then carried out using radiolabeled dNTPs, the oligo-dT-NN anchor primer as 3′ primer and a short arbitrary oligonucleotide as 5′ primer.

Keywords

Polymerase Chain Reaction Polymerase Chain Reaction Product Polymerase Chain Reaction Reaction Amersham Pharmacia Biotech Differential Display 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    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.PubMedCrossRefGoogle Scholar
  2. 2.
    Travis, G. H. and Sutcliffe, J. G. (1988) Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs. Proc. Natl. Acad. Sci. USA 85, 1696–1700.PubMedCrossRefGoogle Scholar
  3. 3.
    Dworkin, M. B. and Dawid, I. B. (1980) Construction of a cloned library of expressed embryonic gene sequences from Xenopus laevis. Dev. Biol. 76, 435–448.PubMedCrossRefGoogle Scholar
  4. 4.
    Lambolez, B., Audinat, E., Bochet, P., Crepel, F., and Rossier, J. (1992) AMPA receptor subunits expressed by single Purkinje cells. Neuron 9, 247–258.PubMedCrossRefGoogle Scholar
  5. 5.
    Liang, P. and Pardee, A. B. (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967–971.PubMedCrossRefGoogle Scholar
  6. 6.
    McClelland, M., Mathieu-Daulde, F., and Welsh, J. (1995) RNA fingerprinting and differential display using arbitrarily primed PCR. Trends Genet 11, 242–246.PubMedCrossRefGoogle Scholar
  7. 7.
    Zimmermann, J. W. and Schultz, R. M. (1994) Analysis of gene expression in the preimplantation mouse embryo: use of mRNA differential display. Proc. Natl. Acad. Sci. USA 91, 5456–5460.PubMedCrossRefGoogle Scholar
  8. 8.
    Li, F., Barnathan, E. S., and Karikó, K. (1994) Rapid method for screening and cloning cDNAs generated in differential mRNA display: application of Northern blot for affinity capturing of cDNAs. Nucl. Acids Res. 22, 1764–1765.PubMedCrossRefGoogle Scholar
  9. 9.
    Watson, M. A. and Fleming, T. P. (1994) Isolation of differentially expressed sequence tags from human breast cancer. Cancer Res. 54, 4598–4602.PubMedGoogle Scholar
  10. 10.
    Wong, K. K. and McClelland, M. (1994) Stress-inducible gene of Salmonella typhimurium identified by arbitrarily primed PCR of RNA. Proc. Natl. Acad. Sci. USA 91, 639–643.PubMedCrossRefGoogle Scholar
  11. 11.
    Liang, P., Averboukh L., Keyomarsi K., Sager R., and Pardee A. B. (1992) Differential display and cloning of messenger RNAs from human breast cancer versus mammary epithelial cells. Cancer Res. 52, 6966–6968.PubMedGoogle Scholar
  12. 12.
    Bauer, D., Muller, H., Reich, J., Riedel, H., Ahrenkiel, V., Warthoe, P., and Strauss, M. (1993) Identification of differentially expressed mRNA species by an improved display technique (DDRT-PCR). Nucl. Acid Res. 21, 4272–4280.CrossRefGoogle Scholar
  13. 13.
    Lohmann, J., Schickle, H. P., and Bosch, T. C. G. (1995) REN, a rapid and efficient method for nonradioactive differential display and isolation of rnRNA. BioTechniques 18(2), 200–202.PubMedGoogle Scholar
  14. 14.
    Bassam, B. J., Caetano-Annolées, G., and Gresshoff, P. M. (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Biochem. 196, 80–83.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Thomas C. G. Bosch
    • 1
  • Jan U. Lohmann
    • 2
  1. 1.Institut für Spezielle ZoologieUniversität JenaGermany
  2. 2.Salk InstituteLa Jolla

Personalised recommendations