A cDNA RDA protocol using solid-phase technology suited for analysis in small tissue samples

cDNA representational difference analysis (cDNA RDA) is a PCR-based subtrac tive enrichment procedure for the cloning of differentially expressed genes . In this study, we have further developed the procedure to take advantage of solid-phase technology, and to facilitate the use of RDA when starting m aterial is limited. Several parameters of the PCR-based generation of cDNA representations were investigated, and a solid-phase based purification ste p was introduced to simplify removal of digested adapter-ends and uncleaved fragments. The use of magnetic particles increased the speed of the method , and also eliminated the risk of carry-over contamination between iterativ e steps of subtraction and PCR amplification. The modified protocol was eva luated in monitoring differences in gene expression in (i) a rat system con sisting of livers with and without growth hormone treatment, and in (ii) a human system consisting of normal colon and colon cancer. (C) 2000 Elsevier Science B.V. All rights reserved.