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Differential Display of Eukaryotic mRNA

  • Antonio Tugores
  • Juan Carlos Izpisua Belmonte
Part of the METHODS IN MOLECULAR BIOLOGY™ book series (MIMB, volume 461)

1. Introduction

Arbitrarily primed PCR is a method that was initially developed to analyze and compare genome complexity (1,2). By using arbitrary primers, an array of stochastic sequences can be amplified using the polymerase chain reaction (PCR) and resolved by denaturing acrylamide gels, generating a characteristic pattern or fingerprint. This technique can be readily applied for the analysis of differentially expressed genes in two different cell types by simply converting the mRNA into cDNA (3,4). A different fingerprint pattern will then reflect differential gene expression between the cell types analyzed. The number and intensity of the fingerprints depend largely on two parameters: the abundance of the original RNAs, and how well the primers match the primary sequence so that efficient amplification can take place. Taking this into consideration, the method appears to be more suitable for the comparison of cell types where a large number of differentially expressed genes are...

Keywords

Sodium Dodecyl Sulfate Polymerase Chain Reaction Product Xylene Cyanol Ethidium Bromide Solution Polymerase Chain Reaction Tube 
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.

Notes

Acknowledgments

This work was supported by Fundacion Cellex, the G. Harold and Leila Y. Mathers Charitable Foundation, Marato and MEC.

References

  1. 1.
    Welsh, J. and McClelland, M (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18, 7213–7218.CrossRefPubMedGoogle Scholar
  2. 2.
    Williams, J. G., Kubelik, A. R., Livak, K. J., Rafalski, J. A., and Tingey, S. V. (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18, 6531–6535.CrossRefPubMedGoogle Scholar
  3. 3.
    Welsh, J., Chada, K., Dalal, S. S., Ralph, D., Chang, R., and McClelland, M. (1992) Arbitrarily primed PCR fingerprinting of RNA. Nucleic Acids Res. 20, 4965– 4970.CrossRefPubMedGoogle Scholar
  4. 4.
    Liang, P. and Pardee, A. B. (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967–971.CrossRefPubMedGoogle Scholar
  5. 5.
    Welsh, J., Chada, K., Dalal, S. S., Cheng, R., Ralph, D., and McClelland, M. (1992) Arbitrarily primed PCR fingerprinting of RNA. Nucleic Acids Res. 20, 4965–4970.CrossRefPubMedGoogle Scholar
  6. 6.
    Ralph, D., McClelland, M., and Welsh, J. (1993) RNA fingerprinting using arbitrarily primed PCR identifies differentially regulated RNAs in mink lung (My1Lu) cells growth arrested by transforming growth factor b1. Proc.Natl.Acad.Sci. USA 90, 10,710–10,714.Google Scholar
  7. 7.
    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
  8. 8.
    Liang, P., Zhu, W., Zhang, X., Guo, Z., O'Connell, R. P., Auerboukh, L., Wang, F., and Pardee, A. B. (1994) Differential display using one-base anchored oligodT. Nucleic Acids Res. 25, 5763,5764.CrossRefGoogle Scholar
  9. 9.
    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.CrossRefPubMedGoogle Scholar
  10. 10.
    Loh, E. Y., Elliot, J. F., Cwirla, S., Lanier, L. L., and Davis, M. M. (1989) Polymer-ase chain reaction with single-sided specificity: Analysis of T cell receptor d chain. Science 243, 217–220.CrossRefPubMedGoogle Scholar
  11. 11.
    Chomczynski, P. and Sacchi, N. (1987) Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem 162, 156–159.CrossRefPubMedGoogle Scholar
  12. 12.
    Feinberg, A. P. and Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13.CrossRefPubMedGoogle Scholar
  13. 13.
    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. Nucleic Acids Res. 22, 1764,1765.CrossRefPubMedGoogle Scholar
  14. 14.
    Honeycutt, R., Sobral, B. W., and McClelland, M. (1997) Polymerase chain reaction (PCR) detection and quantification using a short PCR product and a synthetic internal positive control. Anal. Biochem. 248, 303–306.CrossRefPubMedGoogle Scholar
  15. 15.
    Sambrook, J., Fritsh, E. F., and Maniatis, T. (eds.) (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  16. 16.
    Guimaraes, M. J., Lee, F., Zlotnik, A., and McClanahan, T. (1995) Differential display by PCR: novel findings and applications. Nucleic Acids Res. 23, 1832,1833.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Antonio Tugores
    • 1
  • Juan Carlos Izpisua Belmonte
    • 1
    • 2
  1. 1.Gene Expression Laboratory, Stem Cell Research Center, and Laboratory of GeneticsThe Salk Institute for Biological StudiesLa JollaUSA
  2. 2.The Center of Regenerative Medicine in BarcelonaBarcelonaSpain

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