Detection by In Situ and Northern Hybridization
  • Alessandra P. Princivalle
  • Rachel M. C. Parker
  • Terri J. Dover
  • Nicholas M. Barnes
Part of the Methods in Molecular Biology™ book series (MIMB, volume 306)


The ability to detect mRNA by either in situ hybridization histochemistry (ISHH), first described in 1969 by Gall and Pardue and John et al. (1 , 2) or Northern hybridization, first described by Alwine et al. (3), has become a very powerful technique in many research areas, including that of receptor research. The applications of these techniques are many and include (1) direct assessment of the presence, distribution, and modulation under different physiological conditions of specific RNA species (4, 5); (2) molecular investigations of potential mRNA splice variants and region-specific heterogeneity in multimeric-receptor subunit potential expression (6, 7); (3) indirect detection of receptor-expression to support the existence of the receptor when highly-selective ligands (see  Chapter 5) or antibodies (see  Chapter 8) are unavailable for receptor localization studies (8); and (4) investigation of molecular changes in pathological states and the possible modes of action of drugs used to treat such conditions (9, 10, 11). Changes at the molecular level to alter mRNA expression represent rapid changes within a cell; therefore, it can be envisaged that such studies on human biopsy and post mortem tissue will lead to an array of important diagnostic tools. Furthermore, the combination of ISHH and immunohistochemistry (see  Chapter 8) offers a powerful strategy to study the co-existence of mRNA and the translated polypeptide product (12), with consistent results from the two approaches allowing greater confidence to be attached to the significance of the findings. Alternatively, the co-localization of one mRNA species with a peptide/protein phenotypically characteristic of a certain cell type allows the putative function of the protein to be proposed (13, 14) which subsequently focuses further investigation.


Sodium Dodecyl Sulfate Northern Hybridization Antisense Probe Deionized Formamide Membrane Blot 
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.


  1. 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.PubMedCrossRefGoogle Scholar
  2. 2.
    John, H. A., Birnstiel, M. L., and Jones, K. W. (1969) RNA-DNA hybrids at the cytological level. Nature 223, 582–587.PubMedCrossRefGoogle Scholar
  3. 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.PubMedCrossRefGoogle Scholar
  4. 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.PubMedCrossRefGoogle Scholar
  5. 5.
    Rabadan-Diehl, C., Lolait, S. J., and Aguilera, G.(1995) Regulation of pituitary vasopressin V1b receptor mRNA during stress in the rat. J. Neuroendocrinology 7, 903–910.CrossRefGoogle Scholar
  6. 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.PubMedGoogle Scholar
  7. 7.
    Rigby, M., Le Bourdelles, B., Heavens, R. P., 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.PubMedCrossRefGoogle Scholar
  8. 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. Pharm. 117, 657–666.Google Scholar
  9. 9.
    Taketazu, F., Kato, M., Gobl, A., 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. Laboratory Investigation 70, 620–630.PubMedGoogle Scholar
  10. 10.
    Harrington, K. A., Augood, S. J., Faull, R. l., 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 uantitative cellular in situ hybridisation study. Brain Research. Mol. Br. Res. 33, 333–342.CrossRefGoogle Scholar
  11. 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. Endocrinology 11, 1–7.CrossRefGoogle Scholar
  12. 12.
    Kia, H. K., Miquel, M. C., McKernan, R. M., et al. (1995) Localization of 5-HT3 receptors in the rat spinal cord: immunohistochemistry and in situ hybridization. Neuroreport. 6, 257–261.PubMedCrossRefGoogle Scholar
  13. 13.
    Noguchi, K., Kawalski, K., Traub, R., Solodkin, A., Iadarola, M. J., and Ruda, M. A. (1991) Dynorphin expression andFos-like immunoreactivity following inflammation induced hyperalgesia are colocalised in spinal cord neurones. Mol. Brain Res. 10, 227–233.PubMedCrossRefGoogle Scholar
  14. 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. Neuroscience 17, 3157–3167.Google Scholar
  15. 15.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. eds. (1989) Molecular Cloning: A Laboratory Manual. 2nd edition. Cold Spring Harbour Laboratory, Cold Spring Harbor, New York.Google Scholar
  16. 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. 17.
    Valentino, K. L., Eberwine, J. H., and Barchas, J. D., eds. (1987) In Situ Hybridisation: Applications to Neurobiology. Oxford University Press, NewYork: pp. 57–58.Google Scholar
  18. 18.
    Wilkinson D. G., ed. (1993) In situ Hybridisation: A Practical Approach. IRL Press Oxford University Press, New York.Google Scholar
  19. 19.
    Princivalle, A. P., Duncan, J. S., Thom, M., and Bowery, N. G.(2003) GABAB 1a, GABAB1b and GABAB 2 mRNA variants expression in hippocampus resected from patients with temporal lobe epilepsy. Neuroscience 122, 975–984.PubMedCrossRefGoogle Scholar
  20. 20.
    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-hydrox-ytryptamine type 3As receptor subunit. Mol. Pharmacol. 48, 1054–1062.PubMedGoogle Scholar
  21. 21.
    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.PubMedCrossRefGoogle Scholar
  22. 22.
    Dopazo, J., Zanders, E., Dragoni, I., Amphlett, G., and Falciani, F. (2001) Methods and approaches in the analysis of gene expression data. J. Immunol. Methods 250, 93–112.PubMedCrossRefGoogle Scholar
  23. 23.
    Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 56–159.CrossRefGoogle Scholar
  24. 24.
    Kaupmann, K., Huggel, K., Heid, J., et al. (1997) Expression cloning of GABAB receptors uncovers similarity to metabotropic glutamate receptors. Nature 368, 239–246.CrossRefGoogle Scholar
  25. 25.
    Battaglia, G., Princivalle, A., Forti, F., Lizier, C., and Zeviani, M.(1997) Expression of SMN gene, the spinal muscolar atrophy determining gene, in the mammalian central nervous system. Hum. Mol. Genet. 6, 1961–1971.PubMedCrossRefGoogle Scholar
  26. 26.
    Barnes, N. M., and Sharp, T.(1999) A review of central 5-HT receptors and their function. Neuropharmacology 38, 1083–1152.PubMedCrossRefGoogle Scholar
  27. 27.
    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.PubMedCrossRefGoogle Scholar
  28. 28.
    Feinberg, A. P., and Vogelstein, B.(1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13.PubMedCrossRefGoogle Scholar
  29. 29.
    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.PubMedCrossRefGoogle Scholar
  30. 30.
    Emson, P. C. (1993) In-situ hybridisation as a methodological tool for the neuro-scientist. TINS 16, 9–16.PubMedGoogle Scholar
  31. 31.
    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.PubMedGoogle Scholar
  32. 32.
    Ratcliff, R. C. (1974) Terminal deoxynucleotydyl transferase. In Boyer, P.D., ed., The Enzymes, 3rd ed., vol. XIV. Academic, New York: pp. 105–118.Google Scholar
  33. 33.
    Angerer, L. M. and Angerer, R. C. (1992) In situ hybridisation to cellular RNA with radiolabelled RNA probes. In Wilkinson, D. G., ed. In Situ Hybridization: A Practical Approach IRL Press, Oxford University Press, Oxford: pp. 15–32.Google Scholar
  34. 34.
    Höltke, H. J., Ankenbauer, W., Mühlegger, K., et al. (1995) The digoxigenin (DIG) system for non-radioactive labelling and detection of nucleic acids—an overview. Cell. Mol. Biol. 41, 883–905.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Alessandra P. Princivalle
    • 1
  • Rachel M. C. Parker
    • 2
  • Terri J. Dover
    • 1
  • Nicholas M. Barnes
    • 3
  1. 1.Cellular and Molecular Neuropharmacology Research Group, Division of Neuroscience, Department of PharmacologyThe Medical School, University of BirminghamBirminghamUK
  2. 2.The British Heart FoundationLondonUK
  3. 3.Cellular and Molecular Neuropharmacology Research Group, Division of Neuroscience, Department of PharmacologyThe Medical School, University of BirminghamBirminghamUK

Personalised recommendations