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

Gilbert D, Erdmann G, Zhang X, Fritzsche A, Demir K, Jaedicke A, Muehlenberg K, Wanker E, Boutros M (2011)

Publication Status: Published

Publication Type: Journal article, Original article

Publication year: 2011

Journal

PLoS ONE Public Library of Science

Publisher: Public Library of Science

Book Volume: 6

Pages Range: e28338

Journal Issue: 12

DOI: 10.1371/journal.pone.0028338

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 with CRIS profile

Daniel Gilbert Lehrstuhl für Medizinische Biotechnologie

Involved external institutions

Deutsches Krebsforschungszentrum (DKFZ) DE Germany (DE) Max-Delbrück-Centrum für Molekulare Medizin / Max Delbrück Center for Molecular Medicine (MDC) Berlin-Buch DE Germany (DE) Ruprecht-Karls-Universität Heidelberg DE Germany (DE)

How to cite

APA:

Gilbert, D., Erdmann, G., Zhang, X., Fritzsche, A., Demir, K., Jaedicke, A.,... Boutros, M. (2011). A novel multiplex cell viability assay for high-throughput RNAi screening. PLoS ONE, 6(12), e28338. https://doi.org/10.1371/journal.pone.0028338

MLA:

Gilbert, Daniel, et al. "A novel multiplex cell viability assay for high-throughput RNAi screening." PLoS ONE 6.12 (2011): e28338.

BibTeX: Download