In Situ Hybridization to Brain Tissue Sections Using Labeled Single-Strand Complementary RNA Probes

  • Giulio M. Pasinetti
Part of the Methods in Molecular Biology™ book series (MIMB, volume 13)

Abstract

Because of the vast organizational complexity of the brain, it is crucial to study gene regulation at the level of individual cells. Most brain regions or individual neurons lying adjacent to each other may contain different neurotransmitters or different receptors, which may differentially regulate the gene of interest. Moreover, the brain expresses a much higher proportion of genes than other tissues, such that there are >50,000 distinct mRNA, whose cellular distribution is unknown. This extreme complexity in biochemical composition means that any study of changes in neuronal gene expression in response to pharmacological manipulations (1) or in response to lesions (2) should be analyzed at the singlecell level for maximal sensitivity to detect changes in heterogeneous cell populations.

Keywords

Hydrolysis Crystallization Dust Hydration Mold 

References

  1. 1.
    Pasinetti, G. M., Morgan, D. G., Johnson, S. A., Millar, S. L., and Finch, C. E. (1990) Tyrosine hydroxylase mRNA concentration in midbrain dopaminergic neurons is differentially regulated by reserpine. J. Neurochem. 55, 1793–1799.PubMedCrossRefGoogle Scholar
  2. 2.
    Pasinetti, G. M., Lerner, S. A., Johnson, S. A., Morgan, D. G., Telford, N. A., and Finch C. E. (1989) Chronic lesions differentially decrease tyrosine hydroxylase messenger RNA in dopaminergic neurons of the substantia nigra. Mol. Brain Res. 5, 203–209.PubMedCrossRefGoogle Scholar
  3. 3.
    Henderson, A. S. (1982) Cytological hybridization to mammalian chromosomes. Int. Rev. Cytol. 76, 1–46.PubMedCrossRefGoogle Scholar
  4. 4.
    Brahic, M., Haase, A. T., and Cash, E. (1984) Simultaneous in situ detection of viral RNA and antigens. Proc. Natl. Acad. Sci. USA 81, 5445–5448.PubMedCrossRefGoogle Scholar
  5. 5.
    Pasinetti, G. M., Morgan, D. C., Johnson, S. A., Lerner, S. P., Myers, M. A., Poirier, J., and Finch, C. E. (1989) Combined in situ hybridization and immunocytochemistry in the assay of pharmacological effects on tyrosine hydroxylase mRNA concentration. Pharmacol Res. 21, 299–311.PubMedCrossRefGoogle Scholar
  6. 6.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laburatury Manual, vol. 3, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  7. 7.
    Melton, D., Krieg, P. A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R. (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12, 7035–7056.PubMedCrossRefGoogle Scholar
  8. 8.
    Berger, S. L. (1987) Quantifying 32P-labeled and unlabeled nucleic acid. Meth. Enzymology 158, 49–54.CrossRefGoogle Scholar
  9. 9.
    Cox, K. H., DeLeon, D. V., Angerer, L. M., and Angerer, R. C. (1984) Detection of mRNA in sea urchin embrios by situ hybridization using asymmetric RNA probes. Develop. Biol. 101, 485–502.PubMedCrossRefGoogle Scholar

Copyright information

© The Humana Press, Totowa, NJ 1992

Authors and Affiliations

  • Giulio M. Pasinetti
    • 1
  1. 1.Ethel Percy Andrus Gerontology CenterUniversity of Southern CalifmiaLos Angeles

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