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

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., Antonarakis, S. E., Gingeras, T. R., Guigo, R. (2008) Efficient targeted transcript discovery via array-based normalization of RACE libraries. Nature Methods, 5 (7). pp. 629-35. ISSN 15487091 (ISSN) (Public Dataset)

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URL: http://www.ncbi.nlm.nih.gov/pubmed/18500348
DOI: 10.1038/nmeth.1216

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.

Item Type: Paper
Additional Information: 1548-7105 (Electronic) Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't PMC2713501
Uncontrolled Keywords: Alternative Splicing Chromosomes, Human, Pair 21/genetics Chromosomes, Human, Pair 22/genetics Cloning, Molecular DNA, Complementary/*genetics Exons Gene Expression Profiling/*methods *Gene Library Genome, Human Humans Molecular Sequence Data Nucleic Acid Amplification Techniques/*methods Oligonucleotide Array Sequence Analysis/methods Protein Isoforms/genetics RNA/*genetics Reverse Transcriptase Polymerase Chain Reaction Transcription, Genetic
Subjects: bioinformatics > genomics and proteomics > analysis and processing > DNA RNA Processing
bioinformatics > genomics and proteomics
bioinformatics > genomics and proteomics > analysis and processing > microarray gene expression processing
?? Q1 ??
bioinformatics > genomics and proteomics > alignment > sequence alignment
CSHL Authors:
Communities: CSHL labs > Gingeras lab
Depositing User: Tom Adams
Date: 2008
Date Deposited: 13 Jul 2011 17:55
Last Modified: 12 Jul 2013 18:28
PMCID: PMC2713501
Related URLs:
Dataset ID:
  • GeneBank: FE530175
  • GeneBank: FE530855
URI: http://repository.cshl.edu/id/eprint/7718

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