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Nucleic Acids Research Advance Access published online on March 28, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm141
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© 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

Multiplexing siRNAs to compress RNAi-based screen size in human cells

Scott E. Martin1, Tamara L. Jones1, Cheryl L. Thomas1,2, Philip L. Lorenzi3, Dac A. Nguyen1, Timothy Runfola1, Michele Gunsior4, John N. Weinstein3, Paul K. Goldsmith4, Eric Lader5, Konrad Huppi1 and Natasha J. Caplen1,*

1Gene Silencing Section, Office of Science and Technology Partnership, OD, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, 2Molecular Target Development Program, CCR, NCI-Frederick, NIH, Frederick, 3Genomics and Bioinformatics Group, Laboratory of Molecular Pharmacology, 4Antibody and Protein Purification Unit, CCR, NCI, NIH, Bethesda and 5Qiagen Inc., Germantown, MD, USA

*To whom correspondence should be addressed. Tel: +1 301 451 1844; Fax: +1 301 594 0345; Email: ncaplen{at}mail.nih.gov

Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) corresponding to multiple gene targets in order to compress RNA interference (RNAi) screen size. Before investigating the practical use of this strategy, we first characterized the gene-specific RNAi induced by a large subset (258 siRNAs, 129 genes) of the entire siRNA library used in this study (~800 siRNAs, ~400 genes). We next demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as when the genes were targeted individually. The entire library was then used in a screen in which randomly multiplexed siRNAs were assayed for their affect on cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. This study suggests that the screening of randomly multiplexed siRNAs may provide an important avenue towards the identification of candidate gene targets for downstream functional analyses and may also be useful for the rapid identification of positive controls for use in novel assay systems. This approach is likely to be especially applicable where assay costs or platform limitations are prohibitive.


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