Abstract
The regulation of myocardial gene expression is highly sensitive to any extracellular or intracellular stimulus that affects contractile function. Subtractive suppression hybridization represents a large-scale, unbiased method for detecting transcriptionally and posttranscriptionally regulated genes, both known and unknown, independently of the prevalence of these transcripts. The strength of subtractive hybridization relies on its unbiased nature and its power to extract even low-abundance transcripts. Therefore, the subtraction experiments can reveal “unexpected” gene profiles and can represent a starting point for the cloning and characterization of novel genes. However, the procedure and the subsequent sequencing of the subtracted genes are labor intensive, and, because the method is purely qualitative, it also requires alternative methods of validation.
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References
Nadal-Ginard, B. and Mahdavi, V. (1989) Molecular basis of cardiac performance: plasticity of the myocardium generated through protein isoform switches. J. Clin. Invest. 84, 1693–1700.
Schneider, M., Roberts, R., and Parker, T. (1991) Modulation of cardiac genes by mechanical stress. The oncogene signalling hypothesis. Mol. Biol. Med. 8, 167–183.
Komuro, I. and Yazaki, Y. (1993) Control of cardiac gene expression by mechanical stress. Annu. Rev. Physiol. 55, 55–75.
Komuro, I., Kaida, T., Shibazaki, Y., Kurabayashi, M., Katoh, Y., and Yazaki, Y. (1990) Stretching cardiac myocyte stimulates proto-oncogene expression. J. Biol. Chem. 265, 5391–5398.
Chien, K. R., Zhu, H., Knowlton, K. U., et al. (1993) Transcriptional regulation during cardiac growth and development. Annu. Rev. Physiol. 55, 77–95.
Chien, K., Knowlton, K., Zhu, H., and Chien, S. (1992) Regulation of cardiac gene expression during myocardial growth and hypertrophy: Molecular studies of an adaptive physiologic response. FASEB J. 5, 3037–3046.
Robbins, J. (1996) Regulation of cardiac gene expression during development. Cardiovasc. Res. 31, E2–E16.
Sadoshima, S. and Izumo, S. (1997) The cellular and molecular response of cardiac myocytes to mechanical stress. Annu. Rev. Physiol. 59, 551–571.
Schoenfeld, J., Vasser, M., Jhurani, P., et al. (1998) Distinct molecular phenotypes in murine muscle development, growth and hypertrophy. J. Mol. Cell Cardiol. 30, 2269–2280.
Diatchenko, L., Lau, Y.-F. C., Campbell, A. P., et al. (1996) Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. USA 93, 6025–6030.
Depre, C., Tomlinson, J. E., Kudej, R. K., et al. (2001) Gene program for cardiac cell survival induced by transient ischemia in conscious pig. Proc. Natl. Acad. Sci. USA 98, 9336–9341.
Depre, C., Hase, M., Gaussin, V., et al. (2002) H11 Kinase is a novel mediator of myocardial hypertrophy in vivo. Circ. Res. 91, 1007–1014.
Depre, C., Wang, L., Tomlinson, J., et al. (2003) Characterization of pDJA1, a cardiac-specific chaperone found by genomic profiling of the post-ischemic swine heart. Cardiovasc. Res. 58, 126–135.
Hubank, M. and Schatz, D. (1994) Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res. 22, 5640–5648.
Sehl, P., Tai, J., Hillan, K., et al. (2000) Application of cDNA microarrays in determining molecular phenotype in cardiac growth, development, and response to injury. Circulation 101, 1990–1999.
Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 159–169.
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Depre, C. (2007). Functional Genomics by cDNA Subtractive Hybridization. In: Zhang, J., Rokosh, G. (eds) Cardiac Gene Expression. Methods in Molecular Biology, vol 366. Humana Press. https://doi.org/10.1007/978-1-59745-030-0_4
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DOI: https://doi.org/10.1007/978-1-59745-030-0_4
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