Abstract
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 (9–11). 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.
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References
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.
John, H. A., Birnstiel, M. L., and Jones, K. W. (1969) RNA-DNA hybrids at the cytological level. Nature 223, 582–587.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Hames, B. D. and Higgins, S. J., eds. (1987) Nucleic Acid Hybridisation A Practical Approach. IRL, Oxford University Press, Oxford, UK.
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.
Wilkinson D. G., ed. (1993) In situ Hybridisation: A Practical Approach. IRL Press Oxford University Press, New York.
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.
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.
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.
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.
Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 56–159.
Kaupmann, K., Huggel, K., Heid, J., et al. (1997) Expression cloning of GABAB receptors uncovers similarity to metabotropic glutamate receptors. Nature 368, 239–246.
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.
Barnes, N. M., and Sharp, T.(1999) A review of central 5-HT receptors and their function. Neuropharmacology 38, 1083–1152.
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.
Feinberg, A. P., and Vogelstein, B.(1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13.
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.
Emson, P. C. (1993) In-situ hybridisation as a methodological tool for the neuro-scientist. TINS 16, 9–16.
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.
Ratcliff, R. C. (1974) Terminal deoxynucleotydyl transferase. In Boyer, P.D., ed., The Enzymes, 3rd ed., vol. XIV. Academic, New York: pp. 105–118.
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.
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.
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Princivalle, A.P., Parker, R.M.C., Dover, T.J., Barnes, N.M. (2005). mRNA. In: Davenport, A.P. (eds) Receptor Binding Techniques. Methods in Molecular Biology™, vol 306. Humana Press. https://doi.org/10.1385/1-59259-927-3:051
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DOI: https://doi.org/10.1385/1-59259-927-3:051
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