RNAi microarray analysis in cultured mammalian cells

Spyro Mousses (Corresponding Author), Natasha J. Caplen, Robert Cornelison, Don Weaver, Mark Basik, Sampsa Hautaniemi, Abdel G. Elkahloun, Roberto A. Lotufo, Ashish Choudary, Edward R. Dougherty, Ed Suh, Olli Kallioniemi

Research output: Contribution to journalArticleScientificpeer-review

168 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)2341-2347
JournalGenome Research
Volume13
DOIs
Publication statusPublished - 2003
MoE publication typeA1 Journal article-refereed

Fingerprint

Microarray Analysis
RNA Interference
Cultured Cells
Small Interfering RNA
Transfection
Management Information Systems
Data Display
Gene Knockdown Techniques
Gene Silencing
Genomics
HeLa Cells
Glass
Flow Cytometry
Fluorescence
Technology
Phenotype
Genes

Cite this

Mousses, S., Caplen, N. J., Cornelison, R., Weaver, D., Basik, M., Hautaniemi, S., ... Kallioniemi, O. (2003). RNAi microarray analysis in cultured mammalian cells. Genome Research, 13, 2341-2347. https://doi.org/10.1101/gr.1478703
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. / RNAi microarray analysis in cultured mammalian cells. In: Genome Research. 2003 ; Vol. 13. pp. 2341-2347.
@article{3e86ca99d84e4ce492b964f5b70e537d,
title = "RNAi microarray analysis in cultured mammalian cells",
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.",
author = "Spyro Mousses and Caplen, {Natasha J.} and Robert Cornelison and Don Weaver and Mark Basik and Sampsa Hautaniemi and Elkahloun, {Abdel G.} and Lotufo, {Roberto A.} and Ashish Choudary and Dougherty, {Edward R.} and Ed Suh and Olli Kallioniemi",
year = "2003",
doi = "10.1101/gr.1478703",
language = "English",
volume = "13",
pages = "2341--2347",
journal = "Genome Research",
issn = "1088-9051",
publisher = "Cold Spring Harbor Laboratory Press",

}

Mousses, S, Caplen, NJ, Cornelison, R, Weaver, D, Basik, M, Hautaniemi, S, Elkahloun, AG, Lotufo, RA, Choudary, A, Dougherty, ER, Suh, E & Kallioniemi, O 2003, 'RNAi microarray analysis in cultured mammalian cells', Genome Research, vol. 13, pp. 2341-2347. https://doi.org/10.1101/gr.1478703

RNAi microarray analysis in cultured mammalian cells. / Mousses, Spyro (Corresponding Author); 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.

In: Genome Research, Vol. 13, 2003, p. 2341-2347.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - RNAi microarray analysis in cultured mammalian cells

AU - Mousses, Spyro

AU - Caplen, Natasha J.

AU - Cornelison, Robert

AU - Weaver, Don

AU - Basik, Mark

AU - Hautaniemi, Sampsa

AU - Elkahloun, Abdel G.

AU - Lotufo, Roberto A.

AU - Choudary, Ashish

AU - Dougherty, Edward R.

AU - Suh, Ed

AU - Kallioniemi, Olli

PY - 2003

Y1 - 2003

N2 - 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.

AB - 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.

U2 - 10.1101/gr.1478703

DO - 10.1101/gr.1478703

M3 - Article

VL - 13

SP - 2341

EP - 2347

JO - Genome Research

JF - Genome Research

SN - 1088-9051

ER -

Mousses S, Caplen NJ, Cornelison R, Weaver D, Basik M, Hautaniemi S et al. RNAi microarray analysis in cultured mammalian cells. Genome Research. 2003;13:2341-2347. https://doi.org/10.1101/gr.1478703