ISOLATION OF DIFFERENTIALLY EXPRESSED GENES BY COMBINING REPRESENTATIONAL DIFFERENCE ANALYSIS (RDA) AND CDNA LIBRARY ARRAYS

The difference products (DP) of representational difference analyses ( RDA) were used as hybridization probes on cDNA arrays. The effectivity of RDA products obtained with increasing driver/tester ratios (DP 1 = 100:1, DP 2 = 800:1 and DP 3 = 400 000:1) to isolate differentially e xpressed genes was compared with the effectivity of conventional diffe rential hybridizations. Pacreatic cancer and control tissues were used as a test system to isolate differentially expressed genes. The use o f RDA products as hybridization probes showed two major advantages: (i ) a reliable identification of true differential signals; and (ii) onl y one autoradiograph had to be analyzed, which eliminated the need for a laborious subtraction of signal intensities obtained with different cDNA probes. Increasing driver/tester ratios in iterative rounds of R DA delivered more specific results, though the total yield of differen tial clones was gradually reduced. In this situation, the intermediate RDA product DP 2 provided the best compromise.