RNAi microarray analysis in cultured mammalian cells.
Genome Res
; 13(10): 2341-7, 2003 Oct.
Article
en En
| MEDLINE
| ID: mdl-14525932
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.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Análisis de Secuencia por Matrices de Oligonucleótidos
/
Perfilación de la Expresión Génica
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ARN Interferente Pequeño
Límite:
Humans
Idioma:
En
Año:
2003
Tipo del documento:
Article