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A novel multiplex cell viability assay for high-throughput RNAi screening

Item Type:Article
Title:A novel multiplex cell viability assay for high-throughput RNAi screening
Creators Name:Gilbert, D.F. and Erdmann, G. and Zhang, X. and Fritzsche, A. and Demir, K. and Jaedicke, A. and Muehlenberg, K. and Wanker, E.E. and Boutros, M.
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.
Keywords:Adenosine Triphosphate, Benzimidazoles, Cell Survival, Cytoplasm, False Positive Reactions, Fluoresceins, Fluorescent Dyes, HEK293 Cells, Phenotype, RNA Interference, Reproducibility of Results, Research Design, Small Interfering RNA
Source:PLoS ONE
Publisher:Public Library of Science
Page Range:e28338
Date:5 December 2011
Official Publication:https://doi.org/10.1371/journal.pone.0028338
PubMed:View item in PubMed

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