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Using Multiple Phenotype Assays and Epistasis Testing to Enhance the Reliability of RNAi Screening and Identify Regulators of Muscle Protein Degradation

1
Medical Resarch Council/Arthritis Research UK Centre for Musculoskeletal Ageing Research, University of Nottingham, Royal Derby Hospital, Derby, DE22 3DT, UK
2
Medical Resarch Council/Arthritis Research UK Centre for Musculoskeletal Ageing Research, University of Nottingham, School of Veterinary Medicine & Science, Sutton Bonington, LE12 5RD, UK
3
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA
*
Authors to whom correspondence should be addressed.
Genes 2012, 3(4), 686-701; https://doi.org/10.3390/genes3040686
Received: 13 August 2012 / Revised: 27 September 2012 / Accepted: 30 October 2012 / Published: 2 November 2012
(This article belongs to the Special Issue RNA Interference)
RNAi is a convenient, widely used tool for screening for genes of interest. We have recently used this technology to screen roughly 750 candidate genes, in C. elegans, for potential roles in regulating muscle protein degradation in vivo. To maximize confidence and assess reproducibility, we have only used previously validated RNAi constructs and have included time courses and replicates. To maximize mechanistic understanding, we have examined multiple sub-cellular phenotypes in multiple compartments in muscle. We have also tested knockdowns of putative regulators of degradation in the context of mutations or drugs that were previously shown to inhibit protein degradation by diverse mechanisms. Here we discuss how assaying multiple phenotypes, multiplexing RNAi screens with use of mutations and drugs, and use of bioinformatics can provide more data on rates of potential false positives and negatives as well as more mechanistic insight than simple RNAi screening. View Full-Text
Keywords: RNAi; systems biology; network biology; functional genomics; muscle; proteolysis; C. elegans RNAi; systems biology; network biology; functional genomics; muscle; proteolysis; C. elegans
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Lehmann, S.; Shephard, F.; Jacobson, L.A.; Szewczyk, N.J. Using Multiple Phenotype Assays and Epistasis Testing to Enhance the Reliability of RNAi Screening and Identify Regulators of Muscle Protein Degradation. Genes 2012, 3, 686-701.

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