RNAi microarray analysis in cultured mammalian cells

• Published in: Genome research 2003-10, Vol.13 (10), p.2341-2347
• Main Authors: Mousses, Spyro, Caplen, Natasha J, Cornelison, Robert, Weaver, Don, Basik, Mark, Hautaniemi, Sampsa, Elkahloun, Abdel G, Lotufo, Roberto A, Choudary, Ashish, Dougherty, Edward R, Suh, Ed, Kallioniemi, Olli
• Format: Article
• Language: English
• Subjects: Cell Line, Tumor; DNA microarrays; Gene Expression Profiling - methods; Gene Expression Regulation, Neoplastic - genetics; Green Fluorescent Proteins; HeLa Cells; Humans; Luminescent Proteins - biosynthesis; Luminescent Proteins - genetics; Methods; Oligonucleotide Array Sequence Analysis - methods; RNA Interference; RNA Probes - genetics; RNA, Small Interfering - genetics; Transfection - methods
• ISSN: 1088-9051; 1054-9803
• DOI: 10.1101/gr.1478703

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.