Efficient targeted transcript discovery via array-based normalization of RACE libraries.

TitleEfficient targeted transcript discovery via array-based normalization of RACE libraries.
Publication TypeJournal Article
Year of Publication2008
AuthorsDjebali S, Kapranov P, Foissac S, Lagarde J, Reymond A, Ucla C, Wyss C, Drenkow J, Dumais E, Murray RR, Lin C, Szeto D, Denoeud F, Calvo M, Frankish A, Harrow J, Makrythanasis P, Vidal M, Salehi-Ashtiani K, Antonarakis SE, Gingeras TR, Guigó R
JournalNat. Methods
Volume5
Pagination629–635
Date PublishedJul
Abstract

Rapid amplification of cDNA ends (RACE) is a widely used approach for transcript identification. Random clone selection from the RACE mixture, however, is an ineffective sampling strategy if the dynamic range of transcript abundances is large. To improve sampling efficiency of human transcripts, we hybridized the products of the RACE reaction onto tiling arrays and used the detected exons to delineate a series of reverse-transcriptase (RT)-PCRs, through which the original RACE transcript population was segregated into simpler transcript populations. We independently cloned the products and sequenced randomly selected clones. This approach, RACEarray, is superior to direct cloning and sequencing of RACE products because it specifically targets new transcripts and often results in overall normalization of transcript abundance. We show theoretically and experimentally that this strategy leads indeed to efficient sampling of new transcripts, and we investigated multiplexing the strategy by pooling RACE reactions from multiple interrogated loci before hybridization.

URLhttp://dx.doi.org/10.1038/nmeth.121
DOI10.1038/nmeth.1216