RNAi microarray analysis in cultured mammalian cells.
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.
Mousses
Spyro
S
Cancer Drug Development Laboratory, Translational Genomics Research Institute (TGen), Gaithersburg, Maryland 20878, USA. smousses@tgen.org
Caplen
Natasha J
NJ
Cornelison
Robert
R
Weaver
Don
D
Basik
Mark
M
Hautaniemi
Sampsa
S
Elkahloun
Abdel G
AG
Lotufo
Roberto A
RA
Choudary
Ashish
A
Dougherty
Edward R
ER
Suh
Ed
E
Kallioniemi
Olli
O
D045744:Cell Line, Tumor; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D034622:RNA Interference; D015347:RNA Probes / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection / Q000379:methods
D008164:Luminescent Proteins; D015347:RNA Probes; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins