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

Martin, S.E., Jones, T.L., Thomas, C.L., Lorenzi, P.L., Nguyen, D.A., Runfola, T., Gunsior, M., Weinstein, J.N., Goldsmith, P.K., Lader, E., Huppi, K., Caplen, N.J.
Journal   Nucleic Acids Res.
Species  
Analytes Measured   BAD , EGFR , HIF-1alpha , MEK2 , Met , p53
Matrix Tested   MDA-MB-231 cell lysates, HCT-116 cell lysates
Year   2007
Volume   35
Page Numbers  
Application   Phosphoproteins
Abstract
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 ( approximately 800 siRNAs, approximately 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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