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

Sarah Djebali, Philipp Kapranov, Sylvain Foissac, Julien Lagarde, Alexandre Reymond, Catherine Ucla, Carine Wyss, Jorg Drenkow, Erica Dumais, Ryan R. Murray, Chenwei Lin, David Szeto, France Denoeud, Miquel Calvo, Adam Frankish, Jennifer Harrow, Periklis Makrythanasis, Marc Vidal, Kourosh Salehi-Ashtiani, Stylianos E. AntonarakisThomas R. Gingeras, Roderic Guigó

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)629-635
Number of pages7
JournalNature methods
Volume5
Issue number7
DOIs
StatePublished - Jul 1 2008

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ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Djebali, S., Kapranov, P., Foissac, S., Lagarde, J., Reymond, A., Ucla, C., Wyss, C., Drenkow, J., Dumais, E., Murray, R. R., Lin, C., Szeto, D., Denoeud, F., Calvo, M., Frankish, A., Harrow, J., Makrythanasis, P., Vidal, M., Salehi-Ashtiani, K., ... Guigó, R. (2008). Efficient targeted transcript discovery via array-based normalization of RACE libraries. Nature methods, 5(7), 629-635. https://doi.org/10.1038/nmeth.1216