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

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.

The project at IMIM, CRG, the Universities of Lausanne and Geneva, and Affymetrix is supported by grants U01HG003150 and U01HG003147 from the National Human Genome Research Institute, NIH. Institut Municipal d’Investigació Mèdica and Center for Genomic Regulation (CRG) have also been funded by grant BIO2006-03380 from the Spanish Ministry of Education and Science and from the European BioSapiens Consortium. Affymetrix has also received funds from the National Cancer Institute, NIH, under contract no. N01-CO-12400 and by Affymetrix, Inc. The portion of this work carried out at Center for Cancer Systems Biology was funded by a grant from the Ellison Foundation (awarded to MV) and as Institute Sponsored Research from the Dana Farber Cancer Institute Strategic Initiative