a novel multiplex cell viability assay for high-throughput rnai screening一种新型多路复用单元高通量rnai筛查的可行性分析.pdfVIP

A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening 1 1 1 1 1 1 Daniel F. Gilbert *, Gerrit Erdmann , Xian Zhang , Anja Fritzsche , Kubilay Demir , Andreas Jaedicke , 2 2 1 Katja Muehlenberg , Erich E. Wanker , Michael Boutros 1 German Cancer Research Center (DKFZ), Division of Signaling and Functional Genomics and Heidelberg University, Department of Cell and Molecular Biology, Heidelberg, Germany, 2 Max Delbrueck Center for Molecular Medicine (MDC), Proteomics and Molecular Mechanisms of Neurodegenerative Diseases, Berlin, Germany Abstract Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7 62.3%–58.7614.4% when two indicators were combined and 4