Nucleic Acids Research Advance Access originally published online on November 13, 2007
Nucleic Acids Research 2007 35(21):e146; doi:10.1093/nar/gkm989
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Nucleic Acids Research, 2007, Vol. 35, No. 21 e146
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
A sensitive procedure to detect alternatively spliced mRNA in pooled-tissue samples
1Department of Genetics and Center for Genome Sciences, Washington University in St Louis, 4444 Forest Park Parkway, Campus Box 8510, St Louis, MO 63108, USA and 2WWTF Chair of Bioinformatics, Boku University Vienna, 1190 Muthgasse 18, Vienna, Austria
*To whom the correspondences should be addressed. Tel: +(314)362-2751; Fax: (314)362-2156; Email: rmitra{at}genetics.wustl.edu
Received May 22, 2007. Revised October 22, 2007. Accepted October 22, 2007.
One important goal of genomics is to explore the extent of alternative splicing in the transcriptome and generate a comprehensive catalog of splice forms. New computational and experimental approaches have led to an increase in the number of predicted alternatively spliced transcripts; however, validation of these predictions has not kept pace. In this work, we systematically explore different methods for the validation of cassette exons predicted by computational methods or tiling microarrays. Our goal was to find a procedure that is cost effective, sensitive and specific. We examined three ways of priming the reverse transcription (RT) reaction—poly-dT priming, random priming and pooled exon-specific priming. We also examined two strategies for PCR amplification—flanking PCR, which uses primers that hybridize to the constitutive exons flanking the predicted exon, and a semi-nested PCR with a primer that targets the predicted exon. We found that the combination of RT using a pool of gene-specific primers followed by semi-nested PCR resulted in a significant increase in sensitivity over the most commonly used methodology (97% of the test set was detected versus 14%). Our method was also highly specific—no false positives were detected using a test set of true negatives. Finally, we demonstrate that this method is able to detect alternative exons with a high sensitivity from whole-organism RNA, allowing all tissues to be sampled in a single experiment. The protocol developed here is an accurate and cost-effective way to validate predictions of alternative splicing.
Present address: Germá n Gastó n Leparc, WWTF Chair of Bioinformatics, Boku University Vienna, 1190 Muthgasse 18, Austria