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mRNA: Detection by In Situ and Northern Hybridization

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 106))

Abstract

In situ hybridization histochemistry (ISHH), first described in 1969 by Gall and Pardue and John et al. (1,2) and northern hybridization, first described by Alwine et al. (3), have become very powerful and now quite well established techniques in many research areas, including that of receptor research. Such applications include

  1. 1.

    direct assessment of the presence, distribution, and modulation of specific RNA species under different physiological conditions (4,5);

  2. 2.

    molecular investigations of potential mRNA splice variants and region-specific heterogeneity in multimeric-receptor subunit expression (6,7);

  3. 3.

    indirect identification of receptor-expression to add credence to the existence of that receptor when highly selective ligands are unavailable for receptor binding site localization studies (8); and

  4. 4.

    investigation of molecular changes in pathological states and the possible modes of action of drugs used to treat such conditions (911).

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References

  1. Gall, J. G. and Pardue, M. L. (1969) Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc. Natl. Acad Sci. USA 63, 378–383.

    Article  PubMed  CAS  Google Scholar 

  2. John, H. A., Birnstiel, M. L., and Jones, K. W. (1969) RNA-DNA hybrids at the cytological level. Nature 223, 582–587.

    Article  PubMed  CAS  Google Scholar 

  3. Alwine J. C., Kemp, D. J., and Stark, G. R. (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. USA 74, 5350–5354.

    Article  PubMed  CAS  Google Scholar 

  4. Parker, R. M. C., Fleetwood-Walker, S. M., Rosie, R., Munro, F. E., and Mitchell, R. (1993) Inhibition by NK2 but not NK1 antagonists of carrageenan-induced preprodynorphin mRNA expression in rat dorsal horn lamina I neurons. Neuropeptides 25, 213–222.

    Article  PubMed  CAS  Google Scholar 

  5. Rabadan-Diehl, C., Lolait, S. J., and Aguilera, G. (1995) Regulation of pituitary vasopressin V1b receptor mRNA during stress in the rat. J. Neuroendocrinol. 7, 903–910.

    Article  PubMed  CAS  Google Scholar 

  6. Ciossek, T., Millauer, B., and Ullrich, A. (1995) Identification of alternatively spliced mRNAs encoding varients of MDK1, a novel receptor tyrosine kinase expressed in the murine nervous system. Oncogene 10, 97–108.

    PubMed  CAS  Google Scholar 

  7. Rigby, M., Le Bourdelles, B., Heavens, R. P., Kelly, S., Smith, D., Butler, A. et al. (1996) The messenger RNAs for the N-methyl-D-aspartate receptor subunits show region-specific expression of different subunit composition in the human brain. Neuroscience 73, 429–447.

    Article  PubMed  CAS  Google Scholar 

  8. Gustafson, E. L., Durkin, M. M., Bard, J. A., Zgombick, J., and Branchek, T. A. (1996) A receptor autoradiographic and in situ hybridisation analysis of the distribution of the 5-HT7 receptor in rat brain. Br. J. Pharmacol. 117, 657–666.

    PubMed  CAS  Google Scholar 

  9. Taketazu, F., Kato, M., Gobi, A., Ichijo, H., ten Dijke, P., Itoh, J. et al. (1994) Enhanced expression of transforming growth factor-beta s and transforming growth factor-beta type II receptor in the synovial tissues of patients with rheumatoid arthritis. Lab. Invest. 70, 620–630.

    PubMed  CAS  Google Scholar 

  10. Harrington, K. A., Augood, S. J., Faull, R. I., McKenna, P. J., and Emson, P. C. (1995) Dopamine D1 receptor, D2 receptor, proenkephalin A and substance P gene expression in the caudate nucleus of control and schizophrenic tissue: a quantitative cellular in situ hybridisation study. Brain Res. Mol. Br. Res. 33, 333–342.

    Article  CAS  Google Scholar 

  11. Adcock, I. M., Peters, M., Gelder, C., Shirasaki, H., Brown, C. R., and Barnes, P. J. (1993) Increased tachykinin receptor gene expression in asthmatic lung and its modulation by steroids. J. Mol. Endocrinol. 11, 1–7.

    Article  PubMed  CAS  Google Scholar 

  12. Kia, H. K., Miquel, M. C., McKernan, R. M., Laporte, A. M., Lombard, M. C., Bourgoin, S. et al. (1995) Localization of 5-HT3 receptors in the rat spinal cord: immunohistochemistry and in situ hybridization. Neuroreport. 6, 257–261.

    Article  PubMed  CAS  Google Scholar 

  13. Noguchi, K., Kawalski, K., Traub, R., Solodkin, A., Iadarola, M. J., and Ruda, M. A. (1991) Dynorphin expression and Fos-like immunoreactivity following inflamtion induced hyperalgesia are colocalised in spinal cord neurones. Mol. Brain Res. 10, 227–233.

    Article  PubMed  CAS  Google Scholar 

  14. Morales, M. and Bloom, F. E. (1997) The 5-HT3 receptor is present in different subpopulations of GABAergic neurons in the rat telencephalon. J. Neurosci. 17, 3157–3167.

    PubMed  CAS  Google Scholar 

  15. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. (2nd Ed.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  16. Hames, B. D. and Higgins, S. J. (eds.) (1987) Nucleic Acid Hybridisation a Practical Approach. IRL, Oxford University Press, Oxford, UK.

    Google Scholar 

  17. Rigby, P. W. T., Dieckmann, M., Rhodes, C., and Berg, P. (1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237–251.

    Article  PubMed  CAS  Google Scholar 

  18. Feinberg, A. P. and Vogelstein, B. (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13.

    Article  PubMed  CAS  Google Scholar 

  19. Emson, P. C. (1993) In-situ hybridisation as a methodological tool for the neuro-scientist. TINS 16, 9–16.

    PubMed  CAS  Google Scholar 

  20. Mitchell, B. S., Dhami, D., and Schumacher, U. (1992) Review article: in situ hybridisation: a review of methodologies and applications in the biomedical sciences. Med. Lab. Sci. 49, 107–118.

    PubMed  CAS  Google Scholar 

  21. Lewis, M. E., Sherman, T. G., and Watson, S. J. (1985) In situ hybridisation histochemistry with synthetic oligonucleotides: strategies and methods. Peptides 6(Suppl. 2), 75–87.

    Article  PubMed  CAS  Google Scholar 

  22. Ratcliff, R. C. (1974) Terminal deoxynucleotydyl transferase, in The Enzymes, 3rd Ed. vol. XIV (Boyer, P.D., ed.), Academic, New York, pp. 105–118.

    Google Scholar 

  23. Angerer, L. M. and Angerer, R. C. (1992) In situ hybridisation to cellular RNA with radiolabelled RNA probes, in In Situ Hybridization A Practical Approach (Wilkinson, D. G., ed.) IRL, Oxford University Press, Oxford, U.K., pp. 15–32.

    Google Scholar 

  24. Höltke, H. J., Ankenbauer, W., Mühlegger, K., Rein, R., Sagner, G., Seibl, R. et al. (1995) The digoxigenin (DIG) system for non-radioactive labelling and detection of nucleic acids-an overview. Cell. Mol. Biol. 41, 883–905.

    PubMed  Google Scholar 

  25. Valentino, K. L., Eberwine, J. H., Barchas, J. D. (eds.) (1987) In Situ Hybridisation: Applications to Neurobiology. Oxford University Press, New York, pp. 57–58.

    Google Scholar 

  26. Wilkinson D. G. (ed.) (1993) In Situ Hybridisation: A Practical Approach. IRL, Oxford University Press, New York.

    Google Scholar 

  27. Civelli, O., Douglass, J., Goldstein, A., and Herbert, E. (1985) Sequence and expression of the rat prodynorphin gene. Proc. Natl. Acad. Sci. USA 82, 4291–4295.

    Article  PubMed  CAS  Google Scholar 

  28. Belelli, D., Balcarek, J. M., Hope, A. G., Peters, J. A., Lambert, J. J., and Blackburn, T. P. (1995) Cloning and functional expression of a human 5-hydroxy-tryptamine type 3As receptor subunit. Mol. Pharmacol. 48, 1054–1062.

    PubMed  CAS  Google Scholar 

  29. Parker, R. M. C., Barnes, J. M., Ge, J., Barber, P. C., and Barnes, N. M. (1996) Autoradiographic distribution of [3H]-(s)-zacopride-labelled 5-HT3 receptors in human brain. J. Neurol. Sci. 144, 119–127.

    Article  PubMed  CAS  Google Scholar 

  30. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.

    Article  PubMed  CAS  Google Scholar 

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© 1999 Humana Press Inc., Totowa, NJ

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Parker, R.M.C., Barnes, N.M. (1999). mRNA: Detection by In Situ and Northern Hybridization. In: Keen, M. (eds) Receptor Binding Techniques. Methods in Molecular Biology, vol 106. Springer, Totowa, NJ. https://doi.org/10.1385/0-89603-530-1:247

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  • DOI: https://doi.org/10.1385/0-89603-530-1:247

  • Publisher Name: Springer, Totowa, NJ

  • Print ISBN: 978-0-89603-530-0

  • Online ISBN: 978-1-59259-579-2

  • eBook Packages: Springer Protocols

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