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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-Quelen, 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
Issue7
Pagination629-35
Date Published2008 Jul
ISSN1548-7105
KeywordsAlternative Splicing, Chromosomes, Human, Pair 21, Chromosomes, Human, Pair 22, Cloning, Molecular, DNA, Complementary, Exons, Gene Expression Profiling, Gene Library, Genome, Human, Humans, Molecular Sequence Data, Nucleic Acid Amplification Techniques, Oligonucleotide Array Sequence Analysis, Protein Isoforms, Reverse Transcriptase Polymerase Chain Reaction, RNA, Transcription, Genetic
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.

DOI10.1038/nmeth.1216
Alternate JournalNat. Methods
PubMed ID18500348
PubMed Central IDPMC2713501
Grant ListU01 HG003147-02S1 / HG / NHGRI NIH HHS / United States
U01HG003150 / HG / NHGRI NIH HHS / United States
U01 HG003147 / HG / NHGRI NIH HHS / United States
U01 HG003150-02 / HG / NHGRI NIH HHS / United States
U01 HG003147-01 / HG / NHGRI NIH HHS / United States
U01 HG003150-03S2 / HG / NHGRI NIH HHS / United States
U54 HG004557-03 / HG / NHGRI NIH HHS / United States
U01 HG003150-03 / HG / NHGRI NIH HHS / United States
U01 HG003147-02S2 / HG / NHGRI NIH HHS / United States
U01 HG003147-02 / HG / NHGRI NIH HHS / United States
U54 HG004557-02 / HG / NHGRI NIH HHS / United States
U01 HG003150-03S1 / HG / NHGRI NIH HHS / United States
U54 HG004557 / HG / NHGRI NIH HHS / United States
U54 HG004557-02S1 / HG / NHGRI NIH HHS / United States
U01 HG003147-02S3 / HG / NHGRI NIH HHS / United States
U01HG003147 / HG / NHGRI NIH HHS / United States
U54 HG004557-01 / HG / NHGRI NIH HHS / United States
077198 / / Wellcome Trust / United Kingdom
N01CO12400 / CA / NCI NIH HHS / United States
U01 HG003150-01 / HG / NHGRI NIH HHS / United States
N01-CO-12400 / CO / NCI NIH HHS / United States
U54 HG004555 / HG / NHGRI NIH HHS / United States
U01 HG003150 / HG / NHGRI NIH HHS / United States