Abstract
G-protein-coupled receptor mRNAs are expressed at low levels and therefore present a challenge for the study of their sites and levels of expression. In situ hybridization (ISH) and Northern blotting are powerful methods for the localization of mRNAs and the study of regulation of mRNA expression. ISH combines the power of precise cellular localization with the ability to perform semiquantitative analysis of the mRNA level, whereas Northern blotting has the ability to identify genetic splice variants, or to study multiple RNA molecules sequentially in the same tissue samples.
These protocols give step-by-step instructions for the performance of these techniques, and the analysis of the data that can be obtained using them.
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References
Wilkinson, D. G., (1992) In Situ Hybridization: A Practical Approach, Vol. 109. The Practical Approach Series (Hames, B. D., ed.), Oxford University Press, Oxford.
Masumoto, N., Esaki, T., and Sirotnak, F. M. (2001) Additional organizational features of the murine gamma-glutamyl hydrolase gene. Two remotely situated exons within the complement C3 gene locus encode an alternate 5′ end and proximal ORF under the control of a bidirectional promoter. Gene 268, 183–194.
Michel, U., Stringaris, A. K., Nau, R., and Rieckmann, P. (2000) Differential expression of sense and antisense transcripts of the mitochondrial DNA region coding for ATPase 6 in fetal and adult porcine brain: identification of novel unusually assembled mitochondrial RNAs. Biochem. Biophys. Res. Commun. 271, 170–180.
Van Den Eynde, B. J., Gaugler, B., Probst-Kepper, M., et al. (1999) A new antigen recognized by cytolytic T lymphocytes on a human kidney tumor results from reverse strand transcription. J. Exp. Med. 190, 1793–1800.
Mise, N., Goto, Y., Nakajima, N., and Takagi, N. (1999) Molecular cloning of antisense transcripts of the mouse Xist gene. Biochem. Biophys. Res. Commun. 258, 537–541.
Donaldson, L. F., McQueen, D. S., and Seckl, J. R. (1995) Neuropeptide gene expression and capsaicin-sensitive primary afferents: maintenance and spread of adjuvant arthritis in the rat. J. Physiol. 486, 473–482.
Donaldson, L. F., Haskell, C. A., and Hanley, M. R. (2001) Messenger RNA localization and further characterisation of the putative tachykinin receptor NK4 (NK3B). Recept. Channels 7, 259–272.
Donaldson, L. F., Harmar, A. J., McQueen, D. S., and Seckl, J. R. (1992) Increased expression of preprotachykinin, calcitonin gene-related peptide, but not vasoactive intestinal peptide messenger RNA in dorsal root ganglia during the development of adjuvant monoarthritis in the rat. Brain Res. Mol. Brain Res. 16, 143–149.
Meinkoth, J. and Wahl, G. (1984) Hybridization of nucleic acids immobilized on solid supports. Anal. Biochem. 138, 267–284.
Krumlauf, R. (1994) Analysis of gene expression by northern blot. Mol. Biotechnol. 2, 227242.
Hastie, N. D. and Bishop, J. O. (1976) The expression of three abundance classes of messenger RNA in mouse tissues. Cell 9, 761–774.
Moisan, M. P., Seckl, J. R., and Edwards, C. R. (1990) 11-Beta-hydroxysteroid dehydrogenase bioactivity and messenger RNA expression in rat forebrain: localization in hypothalamus, hippocampus, and cortex. Endocrinology 127, 1450–1455.
Angerer, L. M. and Angerer, R. C. (1992). In situ hybridization to cellular RNA with radiolabelled RNA probes, in In Situ Hybridization. A Practical Approach (Wilkinson, D. G., ed.), Oxford University Press, Oxford, pp. 15–32.
Turgeon, J. L., Van Patten, S. M., Shyamala, G., and Waring, D. W. (1999) Steroid regulation of progesterone receptor expression in cultured rat gonadotropes. Endocrinology 140, 2318–2325.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edit., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.
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Donaldson, L.F. (2004). Identification of G-Protein-Coupled Receptor mRNA Expression by Northern Blotting and In Situ Hybridization. In: Willars, G.B., Challiss, R.A.J. (eds) Receptor Signal Transduction Protocols. Methods in Molecular Biology, vol 259. Humana Press. https://doi.org/10.1385/1-59259-754-8:099
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DOI: https://doi.org/10.1385/1-59259-754-8:099
Publisher Name: Humana Press
Print ISBN: 978-1-58829-329-9
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