NDLI: A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening

Content Provider	PubMed Central
Author	Gilbert, Daniel F. Erdmann, Gerrit Zhang, Xian Fritzsche, Anja Demir, Kubilay Jaedicke, Andreas Muehlenberg, Katja Wanker, Erich E. Boutros, Michael
Editor	Abdelhay, Eliana Saul Furquim Werneck
Copyright Year	2011
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±2.3%–58.7±14.4% when two indicators were combined and 40–48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.
Related Links	http://dx.doi.org/10.1371/journal.pone.0028338
Starting Page	28338
File Format	PDF
ISSN	19326203
e-ISSN	19326203
Journal	PLoS ONE
Issue Number	12
Volume Number	6
Language	English
Publisher	Public Library of Science
Publisher Date	2011-12-01
Access Restriction	Open
Rights Holder	Public Library of Science
Subject Keyword	Biochemistry, Genetics and Molecular Biology(all) Agricultural and Biological Sciences(all) Medicine(all) Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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