RaSH, a rapid subtraction hybridization approach for identifying and cloning differentially expressed genes

Abstract

Human melanoma cells growth-arrest irreversibly and terminally differentiate on treatment with a combination of fibroblast interferon and the protein kinase C activator mezerein. This experimental protocol also results in a loss of tumorigenic potential and profound changes in gene expression. Various cloning and cDNA microarray strategies are being used to determine the complete spectrum of gene expression changes underlying these alterations in human melanoma cells. An efficient approach, Rapid Subtraction Hybridization (RaSH), has been developed that is permitting the identification of genes of potential relevance to cancer growth control and terminal cell differentiation. RaSH cDNA libraries are prepared from double-stranded cDNAs that are enzymatically digested into small fragments, ligated to adapters, and PCR amplified followed by incubation of tester and driver PCR fragments. This subtraction hybridization scheme is technically simple and results in the identification of a high proportion of differentially expressed sequences, including known genes and those not described in current DNA databases. The RaSH approach represents an efficient methodology for identifying and cloning genes displaying differential expression that associate with and potentially regulate complex biological processes.cDNA cloning ͉ reverse Northern blotting ͉ melanoma differentiation associated genes ͉ Northern blotting

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“…Our group has been faced with situations requiring extensive troubleshooting, in cloning projects entailing differential screening, similar to the type likely to be faced in the identi®cation of progression and metastasis regulating genes. We have recently reported two novel PCR based differential screening procedures that are likely to ®nd wide applicability for studying tumor aggression and metastasis (Kang et al, 1998;Jiang et al, 2000).…”

“…Rapid subtraction hybridization (RaSH) (Jiang et al, 2000) is an innovative and rapid PCR based subtractive hybridization approach, which in addition to simplifying cDNA subtraction procedures also results in very ef®cient subtraction. This results in the identi®cation of a signi®cantly higher frequency of validated differentially expressed clones than using any other available approach.…”

“…This results in the identi®cation of a signi®cantly higher frequency of validated differentially expressed clones than using any other available approach. When applied to the process of terminal differentiation and reversion of the cancer phenotype in human melanoma cells, 45% of differentially expressed clones identi®ed using RaSH were con®rmed as bona ®de by Northern blotting (Jiang et al, 2000). Figure 4 provides an outline of this approach.…”

“…Figure 4 provides an outline of this approach. Figure 5 documents the high proportion of differentially expressed genes, as monitored by reverse Northern blotting, obtained using RaSH with human melanoma cells induced to terminally differentiate by treatment with recombinant ®broblast interferon (IFN-b) and the protein kinase C activator mezerein (MEZ) (Jiang et al, 2000). For RaSH, two sources of RNA, for example, from metastatic and corresponding non-metastatic cells respectively, are reverse transcribed using a standard protocol to generate double stranded cDNA libraries.…”

“…The differentially expressed subtracted clones are identi®ed and isolated by their selective ability to ligate into a vector through uniquely compatible ends. This method has been used with considerable success to isolate differentially expressed genes in a human melanoma differentiation system (Jiang et al, 2000;Fig. 2.…”