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A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening
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...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3230607/ https://www.ncbi.nlm.nih.gov/pubmed/22162763 http://dx.doi.org/10.1371/journal.pone.0028338 |
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author | Gilbert, Daniel F. Erdmann, Gerrit Zhang, Xian Fritzsche, Anja Demir, Kubilay Jaedicke, Andreas Muehlenberg, Katja Wanker, Erich E. Boutros, Michael |
author_facet | Gilbert, Daniel F. Erdmann, Gerrit Zhang, Xian Fritzsche, Anja Demir, Kubilay Jaedicke, Andreas Muehlenberg, Katja Wanker, Erich E. Boutros, Michael |
author_sort | Gilbert, Daniel F. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3230607 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-32306072011-12-08 A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening Gilbert, Daniel F. Erdmann, Gerrit Zhang, Xian Fritzsche, Anja Demir, Kubilay Jaedicke, Andreas Muehlenberg, Katja Wanker, Erich E. Boutros, Michael PLoS One Research Article 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. Public Library of Science 2011-12-05 /pmc/articles/PMC3230607/ /pubmed/22162763 http://dx.doi.org/10.1371/journal.pone.0028338 Text en Gilbert et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Gilbert, Daniel F. Erdmann, Gerrit Zhang, Xian Fritzsche, Anja Demir, Kubilay Jaedicke, Andreas Muehlenberg, Katja Wanker, Erich E. Boutros, Michael A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening |
title | A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening |
title_full | A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening |
title_fullStr | A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening |
title_full_unstemmed | A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening |
title_short | A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening |
title_sort | novel multiplex cell viability assay for high-throughput rnai screening |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3230607/ https://www.ncbi.nlm.nih.gov/pubmed/22162763 http://dx.doi.org/10.1371/journal.pone.0028338 |
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