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Methods

RNAi Microarray Analysis in Cultured Mammalian Cells

¹ Cancer Drug Development Laboratory, Translational Genomics Research Institute (TGen), Gaithersburg, Maryland 20878, USA ² Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA ³ Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA ⁴ Department of Computer Engineering and Industrial Automation, State University of Campinas (UNICAMP), Campinas 13081-970, Brazil ⁵ Department of Electrical Engineering, Texas A&M University, College Station, Texas 77843, USA ⁶ Center for Information Technology, National Institutes of Health, Bethesda, Maryland 20892, USA ⁷ Medical Biotechnology Group, VTT Technical Research Centre of Finland and University of Turku, FIN-20521 Turku, Finland

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.

⁹ Present address: Institute of Signal Processing, Tampere University of Technology, Tampere, Finland.

¹⁰ Present address: Translational Genomics Research Institute (TGen), Phoenix, AZ 85004, USA.

¹¹ Corresponding author.
E-MAIL smousses{at}tgen.org; FAX (240) 631-1918.

[Supplemental Material is available online at www.genome.org.]

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.1478703.

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