Cloning Differentially Expressed Genes by Using Differential Display and Subtractive Hybridization

  • Jackson S. Wan
  • Mark G. Erlander
Part of the Methods in Molecular Biology book series (MIMB, volume 85)

Abstract

Differential gene expression occurs in all phases of life, including development, maintenance, injury, and death of an organism. Being able to identify these genes will help understand not only gene function but also the underlying molecular mechanisms of a particular biological system. Differential display (DD) (1,2) and subtractive hybridization (SH) (review see refs. 3, 4, 5) are by far the most common methods currently used by investigators for this purpose. However, do these techniques identify both abundant and rare mRNAs? Previous kinetic studies (6,7) indicate that approx 98% of all mRNA species within the cell have a prevalence ranging from 1/10,000 to 1/100,000, and thus are considered rare. Furthermore, the remaining approx 2% of mRNA species (about 200–500) contribute to over 50% of the total mRNA mass (6,7). Therefore, it is relatively easy to identify a few very abundant differentially expressed mRNAs (i.e., using plus/minus screening); but if the quest is to identify the majority of differentially expressed mRNA species, then a method that is not sensitive to mRNA abundance is required. To evaluate how well DD and SH can identify abundant as well as rare differentially expressed mRNAs, we used both of these methods to find differentially expressed mRNAs within HeLa cells in response to interferon-γ. Described below is a summary of our results (see ref. 8 for original report), followed by detailed protocols of DD, SH, and reverse Northern.

Keywords

Vortex Manifold EDTA MgCl2 Gelatin 

References

  1. 1.
    Liang, P. and Pardee, A. B. (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 251, 967–971.CrossRefGoogle Scholar
  2. 2.
    Welsh, J., Chada, K., Dalai, S. S., Cheng, R., Ralph, D., and McClelland, M. (1992) Arbitrarily primed PCR fingerprinting of RNA. Nucleic Acids Res. 20, 4965–4970.PubMedCrossRefGoogle Scholar
  3. 3.
    Travis, G. H., Milner, R. J., and Sutcliffe, J. G. (1989) Preparation and use of subtractive cDNA hybridization probes for cDNA cloning, in Neuromethods, vol. 16, Molecular Neurobiological Techniques (Boulton, A. A., Baker, G. B., and Campagnoni, A. T., eds.) Humana, Clifton, NJ, pp. 49–78.Google Scholar
  4. 4.
    Watson, J. B. and Margulies, J. E. (1993) Differential cDNA screening strategies to identify novel stage-specific proteins in the developing mammalian brain. Developmental Neurosci. 15, 77–86.CrossRefGoogle Scholar
  5. 5.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  6. 6.
    Axel, R., Feigelson, P., and Schutz, G. (1976) Analysis of the complexity and diversity of mRNA from chicken liver and oviduct. Cell 7, 247–254.PubMedCrossRefGoogle Scholar
  7. 7.
    Bishop, J. O., Morton, J. G., Rosbash, M., and Richardson, M. (1974) Three abundance classes in HeLa cell messenger RNA. Nature 250, 199–204.PubMedCrossRefGoogle Scholar
  8. 8.
    Wan, J. S., Sharp, S. J., Poirier, G. M.-C., Wagaman, P. C., Chambers, J., Pyati, J., Horn, Y.-L., Galindo, J. E., Huvar, A., Peterson, P. A., Jackson, M. R., and Erlander, M. G. (1996) Cloning differentially expressed mRNAs. Nature Biotechnol 14, 1685–1691.CrossRefGoogle Scholar
  9. 9.
    Bertioli, D. J., Schlichter, U. H., Adams, M. J., Burrows, P. R., Steinbiss, H. H., and Antoniw, J. F. (1995) An analysis of differential display shows a strong bias towards high copy number mRNAs. Nucleic Acids Res. 23, 4520–4523.PubMedCrossRefGoogle Scholar
  10. 10.
    Debouck, C. (1995) Differential display or differential dismay? Curr. Opinion Biotechnol. 6, 597–599.CrossRefGoogle Scholar
  11. 11.
    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). Nucleic Acids Res. 21, 4272–4280.PubMedCrossRefGoogle Scholar
  12. 12.
    Van Gelder, R. N., von Zastrow, M. E., Yool, A., Dement, W. C., Barchas, J. D., and Eberwine, J. H. (1990) Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc. Natl Acad. Sci. USA 87, 1663–1667.PubMedCrossRefGoogle Scholar
  13. 13.
    Poirier, G. M.-C., Pyati, J., Wan, J. S., and Erland, M. G. (1997) Screening differentially expressed cDNA clones obtained by differential display using amplified RNA. Nucleic Acids Res. 25, 913,914.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1997

Authors and Affiliations

  • Jackson S. Wan
    • 1
  • Mark G. Erlander
    • 1
  1. 1.R. W. Johnson Pharmaceutical Research InstituteSan Diego

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