title: A novel multiplex cell viability assay for high-throughput RNAi screening.
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.
authors.affiliation: German Cancer Research Center (DKFZ), Division of Signaling and Functional Genomics and Heidelberg University, Department of Cell and Molecular Biology, Heidelberg, Germany. daniel.gilbert@mbt.uni-erlangen.de
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Gilbert
authors.forename: Gerrit
authors.initials: G
authors.lastname: Erdmann
authors.forename: Xian
authors.initials: X
authors.lastname: Zhang
authors.forename: Anja
authors.initials: A
authors.lastname: Fritzsche
authors.forename: Kubilay
authors.initials: K
authors.lastname: Demir
authors.forename: Andreas
authors.initials: A
authors.lastname: Jaedicke
authors.forename: Katja
authors.initials: K
authors.lastname: Muehlenberg
authors.forename: Erich E
authors.initials: EE
authors.lastname: Wanker
authors.forename: Michael
authors.initials: M
authors.lastname: Boutros
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D001562:Benzimidazoles / Q000494:pharmacology; D002470:Cell Survival; D003593:Cytoplasm / Q000378:metabolism; D005189:False Positive Reactions; D005452:Fluoresceins / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D010641:Phenotype; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015203:Reproducibility of Results; D012107:Research Design
chemical_list: D001562:Benzimidazoles; D005452:Fluoresceins; D005456:Fluorescent Dyes; D034741:RNA, Small Interfering; C085925:calcein AM; D000255:Adenosine Triphosphate; C017807:bisbenzimide ethoxide trihydrochloride