Cloning Differentially Expressed Genes Using Rapid Subtraction Hybridization (RaSH)
Differential gene expression represents the entry point for comprehending complex biological processes. In this context, identification and cloning of differentially expressed genes represent critical elements in this process. Many techniques have been developed to facilitate achieving these objectives. Although effective in many situations, most currently described approaches are not trouble-free and have limitations, including complexity of performance, redundancy of gene identification (reflecting cloning biases) and false-positive gene identification. A detailed methodology to perform a rapid and efficient cloning approach, called rapid subtraction hybridization is described in this chapter. This strategy has been applied successfully to a number of cell culture systems and biological processes, including terminal differentiation and cancer progression in human melanoma cells, resistance or sensitivity to HIV-1 in human T cells and gene expression changes following infection of normal human fetal astrocytes with HIV-1 or treatment with neutrotoxic agents. Based on its simplicity of performance and high frequency of genuine differential gene identification, the rapid subtraction hybridization (RaSH) approach will allow wide applications in diverse systems and biological contexts.
Key WordsRaSH gene cloning terminal differentiation metastasis reverse Northern Northern blotting
- 13.Ivanova, I. B., Fesenko, I. V., and Beliavskii, A. V. (1994) A new method of comparative analysis of gene expression and identification of differentially expressed mRNA. Mol. Biol. (Mosk) 28, 1367–1375.Google Scholar
- 15.Kang, D.-c., Jiang, H., Su, Z.-z., Volsky, D. J., and Fisher, P. B. (2002) RaSH-Rapid subtraction hybridization, in Analysing Gene Expression (Lorkowski, S., and Cullen, P., eds.), Germany: Wiley-VCH Verlag GmbH, pp. 206–214.Google Scholar
- 16.Kang, D.-c., Su, Z.-z., Boukerche, H., and Fisher, P. B. (2006) Identification of differentially expressed genes using rapid subtraction hybridization (RaSH): detailed methodology for performing RaSH, in Immunohistochemistry and In Situ Hybridization of Human Carcinomas: Molecular Genetics, Liver Carcinoma, and Pancreatic Carcinoma, (Hyat, M. A., ed.), Elsevier/Academic Press, CA, Vol. 3, in press.Google Scholar
- 21.Jiang, H. and Fisher, P. B. (1993) Use a sensitive and efficient subtraction hybridization protocol for the identification of genes differentially regulated during the induction of differentiation in human melanoma cells. Mol. Cell Differ. 1, 285–299.Google Scholar