Nature Methods 3, 385 - 390 (2006)
Published online: 20 April 2006; | doi:10.1038/nmeth876
High-throughput RNAi screening by time-lapse imaging of live human cellsBeate Neumann1, 4, Michael Held1, 4, Urban Liebel1, 4, Holger Erfle1, 4, Phill Rogers1, Rainer Pepperkok2
& Jan Ellenberg2, 31
MitoCheck Project Group, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. 2
Cell Biology/Biophysics, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. 3
Gene Expression Programmes, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. 4
These authors contributed equally to this work.
Correspondence should be addressed to Jan Ellenberg jan.ellenberg@embl.de RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.
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