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

Djebali, Sarah
Kapranov, Philipp
Foissac, Sylvain
Lagarde, Julien
Wyss, Carine
Drenkow, Jorg
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Published in Nature Methods. 2008, vol. 5, no. 7, p. 629-635
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.
Keywords Alternative SplicingChromosomes, Human, Pair 21/geneticsChromosomes, Human, Pair 22/geneticsCloning, MolecularDNA, Complementary/ geneticsExonsGene Expression Profiling/ methodsGene LibraryGenome, HumanHumansMolecular Sequence DataNucleic Acid Amplification Techniques/ methodsOligonucleotide Array Sequence Analysis/methodsProtein Isoforms/geneticsRNA/ geneticsReverse Transcriptase Polymerase Chain ReactionTranscription, Genetic
PMID: 18500348
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DJEBALI, Sarah et al. Efficient targeted transcript discovery via array-based normalization of RACE libraries. In: Nature methods, 2008, vol. 5, n° 7, p. 629-635. doi: 10.1038/nmeth.1216 https://archive-ouverte.unige.ch/unige:9167

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Deposited on : 2010-07-12

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