Recovery of developmentally defined gene sets from high-density cDNA macroarrays.
New technologies for isolating differentially expressed genes from large arrayed cDNA libraries are reported. These methods can be used to identify genes that lie downstream of developmentally important transcription factors and genes that are expressed in specific tissues, processes, or stages of embryonic development. Though developed for the study of gene expression during the early embryogenesis of the sea urchin Strongylocentrotus purpuratus, these technologies can be applied generally. Hybridization parameters were determined for the reaction of complex cDNA probes to cDNA libraries carried on six nylon filters, each containing duplicate spots from 18,432 bacterial clones (macroarrays). These libraries are of sufficient size to include nearly all genes expressed in the embryo. The screening strategy we have devised is designed to overcome inherent sensitivity limitations of macroarray hybridization and thus to isolate differentially expressed genes that are represented only by low-prevalence mRNAs. To this end, we have developed improved methods for the amplification of cDNA from small amounts of tissue (as little as approximately 300 sea urchin embryos, or 2 x 10(5) cells, or about 10 ng of mRNA) and for the differential enhancement of probe sequence concentration by subtractive hybridization. Quantitative analysis of macroarray hybridization shows that these probes now suffice for detection of differentially expressed mRNAs down to a level below five molecules per average embryo cell.
Rast
J P
JP
Division of Biology 156-29, California Institute of Technology, Pasadena, California 91125, USA.
Amore
G
G
Calestani
C
C
Livi
C B
CB
Ransick
A
A
Davidson
E H
EH
D000818:Animals; D030801:Animals, Genetically Modified; D015342:DNA Probes; D018076:DNA, Complementary; D004625:Embryo, Nonmammalian / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics; D012617:Sea Urchins / Q000196:embryology* / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism
D015342:DNA Probes; D018076:DNA, Complementary; D008164:Luminescent Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D049452:Green Fluorescent Proteins