PCR Amplification of Viral DNA and Viral Host Cell mRNAs in Situ

  • Janet Embretson
  • Katherine Staskus
  • Ernest Retzel
  • Ashley T. Haase
  • Peter Bitterman

Abstract

The hallmark and power of in situ hybridization methodology are its ability to determine the levels of gene expression in an individual cell in a population or determine which cells in a population have acquired new genes by infection or other processes. In this way one can gain insight into how levels of gene expression can vary within a population with the same complement of genes, how different genes within a cell are coordinately regulated, and how newly introduced genes are regulated and interact with genes in the host cell. In contrast to other techniques that measure the average number of molecules per cell in the population, in situ analyses measure the number of molecules in a particular cell in a particular spatial and temporal context. With double-labeling techniques, one can also appreciate, for example, the relative abundancy of specific nucleic acid and protein in the same cell.

Keywords

Formaldehyde Electrophoresis Paraffin Paraformaldehyde Xylene 

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References

  1. Bagasra O, Hauptman SP, Lischner HW, Sachs M, Pomerantz RJ (1992): Detection of human immunodeficiency virus type 1 provirus in mononuclear cells by in situ polymerase chain reaction. N Engl J Med 326:1385–1391.PubMedCrossRefGoogle Scholar
  2. Bitterman P, Haase A, Embretson J, Peterson M, Harman K, Jessurum J (1993): Combination of the polymerase chain reaction with in situ hybridization to define the spatial pattern of mRNA in tissue. Manuscript submitted.Google Scholar
  3. Embretson J, Zupanocic M, Beneke J, Till M, Wolinsky S, Ribas JL, Burke A, Haase AT (1993): Analysis of human immunodeficiency virus infected tissues by amplification and in situ hybridization reveals latent and permissive infections at single cell resolution. Proc Natl Acad Sci USA 90:357–361.PubMedCrossRefGoogle Scholar
  4. Fauci AS (1988): The human immunodeficiency virus: Infectivity and mechanisms of pathogenesis. Science 239:617–622.PubMedCrossRefGoogle Scholar
  5. Haase AT, Retzel EF, Staskus KA (1990): Amplification and detection of lentiviral DNA inside cells. Proc Natl Acad Sci USA 87:4971–4975.PubMedCrossRefGoogle Scholar
  6. Harper ME, Marselle LM, Gallo RC, Wong-Staal F (1986): Detection of lymphocytes expressing human T-lymphotropic virus III in lymph nodes and peripheral blood from infected individuals by in situ hybridization. Proc Natl Acad Sci USA 83:722–776.CrossRefGoogle Scholar
  7. Levy JA (1988): Mysteries of HIV: Challenges for therapy and prevention. Nature (London) 333:519–522.CrossRefGoogle Scholar
  8. Nuovo GJ, Gallery F, MacConnell P, Becker J, Bloch W (1991a): An improved technique for the in situ detection of DNA after polymerase chain reaction amplification. Am J Pathol 139:1239–1244.PubMedGoogle Scholar
  9. Nuovo GJ, MacConnell P, Forde A, DeIvenne P (1991b): Detection of human papilloma virus DNA in formalin-fixed tissues by in situ hybridization after amplification by polymerase chain reaction. Am J Pathol 139:847–854.PubMedGoogle Scholar
  10. Staskus KA, Couch L, Bitterman P, Retzel EF, Zupancic M, List JF, Haase AT (1991): In situ amplification of visna virus DNA in tissue sections reveals a reservoir of latently infected cells. Microbial Path 11:67–76.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1994

Authors and Affiliations

  • Janet Embretson
  • Katherine Staskus
  • Ernest Retzel
  • Ashley T. Haase
  • Peter Bitterman

There are no affiliations available

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