Title | Efficient targeted transcript discovery via array-based normalization of RACE libraries. |
Publication Type | Journal Article |
Year of Publication | 2008 |
Authors | Djebali-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 |
Journal | Nat Methods |
Volume | 5 |
Issue | 7 |
Pagination | 629-35 |
Date Published | 2008 Jul |
ISSN | 1548-7105 |
Keywords | Alternative 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. |
DOI | 10.1038/nmeth.1216 |
Alternate Journal | Nat. Methods |
PubMed ID | 18500348 |
PubMed Central ID | PMC2713501 |
Grant List | U01 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 |