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Type: Artigo de periódico
Title: RNAi microarray analysis in cultured mammalian cells
Author: Mousses, S
Caplen, NJ
Cornelison, R
Weaver, D
Basik, M
Hautaniemi, S
Elkahloun, AG
Lotufo, RA
Choudary, A
Dougherty, ER
Suh, E
Kallioniemi, O
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.
Country: EUA
Editor: Cold Spring Harbor Lab Press, Publications Dept
Rights: embargo
Identifier DOI: 10.1101/gr.1478703
Date Issue: 2003
Appears in Collections:Artigos e Materiais de Revistas Científicas - Unicamp

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