Representational difference analysis of cDNA.

In this chapter 1 describe the PCR-coupled subtractive hybridization technique of representational difference analysis of cDNA (cDNA RDA). cDNA RDA is based on the representational difference analysis (RDA) method previously described by Lisitsyn et al., and can be used to identify genes whose expression is modified between two populations of cells. cDNA RDA is relatively inexpensive to perform and requires no prior knowledge of genome sequence data. The combining of PCR with a subtractive methodology results in a highly effective and extremely sensitive technique with application to very low amounts of starting material. The procedure can be divided into three main phases: PCR generation of amplicons representative of the starting populations of RNA molecules being compared; the two-step subtractive hybridization of these representations, leading to the enrichment of amplified fragments of differentially expressed genes and the sequential depletion of sequences common to both populations; and the purification, cloning, and sequencing of the resulting difference products.