Recovery of developmentally defined gene sets from high-density cDNA macroarrays

New technologies for isolating differentially expressed genes from large ar rayed cDNA libraries are reported. These methods can be used to identify ge nes that lie downstream of developmentally important transcription factors and genes that are expressed in specific tissues, processes, or stages of e mbryonic development. Though developed for the study of gene expression dur ing the early embryogenesis of the sea urchin Strongylocentrotus purpuratus , these technologies can be applied generally. Hybridization parameters wer e determined for the reaction of complex cDNA probes to cDNA libraries carr ied on six nylon filters, each containing duplicate spots from 18,432 bacte rial clones (macroarrays). These libraries are of sufficient size to includ e nearly all genes expressed in the embryo. The screening strategy we have devised is designed to overcome inherent sensitivity limitations of macroar ray hybridization and thus to isolate differentially expressed genes that a re represented only by low-prevalence mRNAs. To this end, we have developed improved methods for the amplification of cDNA from small amounts of tissu e (as little as similar to 300 sea urchin embryos, or 2 x 10(5) cells, or a bout 10 ng of mRNA) and for the differential enhancement of probe sequence concentration by subtractive hybridization. Quantitative analysis of macroa rray hybridization shows that these probes now suffice for detection of dif ferentially expressed mRNAs down to a level below five molecules per averag e embryo cell. (C) 2000 Academic Press.