IDENTIFICATION OF GENES WITH SPECIFIC EXPRESSION IN PANCREATIC-CANCERBY CDNA REPRESENTATIONAL DIFFERENCE ANALYSIS

cDNA representational difference analysis (cDNA-RDA) is a polymerase-c hain-reaction-coupled subtractive and kinetic enrichment procedure for the isolation of differentially expressed genes. In this study, the t echnique was used to isolate novel genes specifically expressed in pan creatic cancer. cDNA-RDA was done on cDNA reverse transcribed from a p oly(A)(+) mRNA pool made from 10 cancer tissues (tester) by using as a driver a cDNA from a poly(A)(+) mRNA pool made from a combination of 10 tissues of chronic pancreatitis and 10 healthy pancreatic tissues. The use of chronic pancreatitis in addition to healthy pancreas mRNA i n the driver preparation eliminated the influence of stromal tissue co mponents present as contamination in the cancer-specific preparations. Such cDNA-RDA led to the isolation of 16 distinct, cancer-specific ge ne fragments. These were confirmed to be overexpressed in pancreatic c ancer tissues by Northern blot analysis. Sequence analysis revealed ho mologies to five genes previously implicated in the carcinogenesis of the pancreas or other tissues. Eleven fragments had no significant hom ology to any known gene and thus represent novel candidate disease gen es. The experiments demonstrate that cDNA-RDA is a reproducible and hi ghly efficient method for the identification of novel genes with cance r-specific expression. (C) 1997 Wiley-Liss, Inc.