RNAi microarray analysis in cultured mammalian cells

Spyro Mousses (Corresponding Author), Natasha J. Caplen, Robert Cornelison, Don Weaver, Mark Basik, Sampsa Hautaniemi, Abdel G. Elkahloun, Roberto A. Lotufo, Ashish Choudary, Edward R. Dougherty, Ed Suh, Olli Kallioniemi

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168 Citations (Scopus)

Abstract

RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) is a powerful new tool for analyzing gene knockdown phenotypes in living mammalian cells. To facilitate large-scale, high-throughput functional genomics studies using RNAi, we have developed a microarray-based technology for highly parallel analysis. Specifically, siRNAs in a transfection matrix were first arrayed on glass slides, overlaid with a monolayer of adherent cells, incubated to allow reverse transfection, and assessed for the effects of gene silencing by digital image analysis at a single cell level. Validation experiments with HeLa cells stably expressing GFP showed spatially confined, sequence-specific, time- and dose-dependent inhibition of green fluorescence for those cells growing directly on microspots containing siRNA targeting the GFP sequence. Microarray-based siRNA transfections analyzed with a custom-made quantitative image analysis system produced results that were identical to those from traditional well-based transfection, quantified by flow cytometry. Finally, to integrate experimental details, image analysis, data display, and data archiving, we developed a prototype information management system for high-throughput cell-based analyses. In summary, this RNAi microarray platform, together with ongoing efforts to develop large-scale human siRNA libraries, should facilitate genomic-scale cell-based analyses of gene function.
Original languageEnglish
Pages (from-to)2341-2347
JournalGenome Research
Volume13
DOIs
Publication statusPublished - 2003
MoE publication typeA1 Journal article-refereed

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    Mousses, S., Caplen, N. J., Cornelison, R., Weaver, D., Basik, M., Hautaniemi, S., Elkahloun, A. G., Lotufo, R. A., Choudary, A., Dougherty, E. R., Suh, E., & Kallioniemi, O. (2003). RNAi microarray analysis in cultured mammalian cells. Genome Research, 13, 2341-2347. https://doi.org/10.1101/gr.1478703