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

Lehmann, Susann; Shephard, Freya; Jacobson, Lewis A.; Szewczyk, Nathaniel J.

doi:10.3390/genes3040686

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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

Susann Lehmann

^1,*,

Freya Shephard

²,

Lewis A. Jacobson

³ and

Nathaniel J. Szewczyk

^1,*

Medical Resarch Council/Arthritis Research UK Centre for Musculoskeletal Ageing Research, University of Nottingham, Royal Derby Hospital, Derby, DE22 3DT, UK

Medical Resarch Council/Arthritis Research UK Centre for Musculoskeletal Ageing Research, University of Nottingham, School of Veterinary Medicine & Science, Sutton Bonington, LE12 5RD, UK

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)

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Abstract

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

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MDPI and ACS Style

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. https://doi.org/10.3390/genes3040686

AMA Style

Lehmann S, Shephard F, Jacobson LA, Szewczyk NJ. Using Multiple Phenotype Assays and Epistasis Testing to Enhance the Reliability of RNAi Screening and Identify Regulators of Muscle Protein Degradation. Genes. 2012; 3(4):686-701. https://doi.org/10.3390/genes3040686

Chicago/Turabian Style

Lehmann, Susann, Freya Shephard, Lewis A. Jacobson, and Nathaniel J. Szewczyk. 2012. "Using Multiple Phenotype Assays and Epistasis Testing to Enhance the Reliability of RNAi Screening and Identify Regulators of Muscle Protein Degradation" Genes 3, no. 4: 686-701. https://doi.org/10.3390/genes3040686

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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

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