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Nucleic Acids Research 2006 34(2):e13; doi:10.1093/nar/gnj015
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Published online 23 January 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Methods Online

Thorough validation of siRNA-induced cell death phenotypes defines new anti-apoptotic protein

Weilin Wu*, Emily Hodges and Christer Höög

Center for Genomics and Bioinformatics, Karolinska Institute Stockholm, SE-17177, Sweden

*To whom correspondence should be addressed. Tel: +46 8 5248 7396; Fax: +46 8 32 36 72; Email: weilin.wu{at}cgb.ki.se

Received November 14, 2005. Revised January 11, 2006. Accepted January 11, 2006.

Loss-of-function by means of RNA interference in cultured human cells enables rapid pathway dissection on a genome-scale. Improved siRNA design and key validation protocols are required to eliminate falsely identified phenotypes resulting from potential off-target consequences. Here, we demonstrate a validation strategy involving several steps for verifying cell death phenotypes revealed during loss-of-function screening. First, from a set of 45 novel human genes we identified gene candidates that, when silenced, induce apoptosis in cultured HeLa cells. For those candidates, we performed more extensive validation with multiple effective siRNAs. In addition, we designed rescue experiments involving candidate genes delivered exogenously and containing silent mutations in the siRNA target regions. Rescue of the observed knockdown phenotype demonstrated an original and more stringent validation of the siRNA's selectivity and the phenotype specificity for the target gene. As a result, our data reveals an anti-apoptotic function for novel human breast adenocarcinoma marker BC-2, adding new depth to BC-2's description as a putative tumor marker involved in cancer related pathways.


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