Efficient targeted transcript discovery via array-based normalization of RACE libraries.
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.
Djebali
Sarah
S
Grup de Recerca en Informàtica Biomèdica, Institut Municipal d'Investigació Mèdica/Universitat Pompeu Fabra, Dr. Aiguader 88, 08003 Barcelona, Spain.
Kapranov
Philipp
P
Foissac
Sylvain
S
Lagarde
Julien
J
Reymond
Alexandre
A
Ucla
Catherine
C
Wyss
Carine
C
Drenkow
Jorg
J
Dumais
Erica
E
Murray
Ryan R
RR
Lin
Chenwei
C
Szeto
David
D
Denoeud
France
F
Calvo
Miquel
M
Frankish
Adam
A
Harrow
Jennifer
J
Makrythanasis
Periklis
P
Vidal
Marc
M
Salehi-Ashtiani
Kourosh
K
Antonarakis
Stylianos E
SE
Gingeras
Thomas R
TR
Guigó
Roderic
R
D017398:Alternative Splicing; D002891:Chromosomes, Human, Pair 21 / Q000235:genetics; D002892:Chromosomes, Human, Pair 22 / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D005091:Exons; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D020033:Protein Isoforms / Q000235:genetics; D012313:RNA / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D018076:DNA, Complementary; D020033:Protein Isoforms; D012313:RNA