RNAi Microarray Analysis in Cultured Mammalian Cells

  1. Spyro Mousses1,2,8,11,
  2. Natasha J. Caplen3,8,
  3. Robert Cornelison2,
  4. Don Weaver1,2,
  5. Mark Basik1,2,
  6. Sampsa Hautaniemi2,9,
  7. Abdel G. Elkahloun2,
  8. Roberto A. Lotufo4,
  9. Ashish Choudary5,
  10. Edward R. Dougherty5,
  11. Ed Suh6,10, and
  12. Olli Kallioniemi1,2,7
  1. 1 Cancer Drug Development Laboratory, Translational Genomics Research Institute (TGen), Gaithersburg, Maryland 20878, USA
  2. 2 Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA
  3. 3 Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA
  4. 4 Department of Computer Engineering and Industrial Automation, State University of Campinas (UNICAMP), Campinas 13081-970, Brazil
  5. 5 Department of Electrical Engineering, Texas A&M University, College Station, Texas 77843, USA
  6. 6 Center for Information Technology, National Institutes of Health, Bethesda, Maryland 20892, USA
  7. 7 Medical Biotechnology Group, VTT Technical Research Centre of Finland and University of Turku, FIN-20521 Turku, Finland

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.

Footnotes

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

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

  • 8 These authors contributed equally to this work.

  • 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.

    • Accepted August 4, 2003.
    • Received May 19, 2003.
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