Abstract

RNA interference 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. However, 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. All steps including the production of siRNA microarrays, fluorescence imaging and computational phenotyping of digital images were automated in a genome-wide high-throughput workflow. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens. Results of the Mitocheck screen are available at www.mitocheck.org.