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

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.

The integrin‐adhesome is required to maintain muscle structure, mitochondrial ATP production, and movement forces inCaenorhabditis elegans

“…This analysis revealed that 84% of the behavioral phenotypes observed in the present RNAi screen were consistent with past studies, whereas 7% of our observations appear to be the first report of a behavioral phenotype for knockdown of the targeted gene and 10% of our observations do not confirm past reports. These values are similar to other large-scale RNAi screens from our laboratory ( 18 , 28 ) and suggest that the level of variability in RNAi knockdown is roughly 10%, on par with many standard techniques such as Western blotting ( 29 ).…”

A quantitative genome-wide RNAi screen in C. elegans for antifungal innate immunity genes

“…If the clones we have identified are true positives, then these 155 clones should also have been retained. This gives a measure of the efficiency of the screen (360/(360 + 155), 70 %); in common with other RNAi screens (e.g., [ 32 , 33 ]), false negatives are thus a notable limitation here. Among the identified targets, in addition to rack-1 , we noted the presence of nipi-3 , nsy-1 , pkc-3 , sta-2 and tir-1 , all previously characterized for their role in regulating antimicrobial peptide gene expression [ 1 , 3 , 7 , 9 ].…”

Knockdown of the C. elegans Kinome identifies Kinases required for normal protein Homeostasis, Mitochondrial network structure, and Sarcomere structure in muscle

“…Similarly, while our use of RNAi by feeding has allowed us to define a preliminary in vivo functional kinome requirement for muscle, our use of this single method will almost certainly have caused us to overlook certain kinases. For example, RNAi by feeding does not produce a reproducible quantitative knockdown from animal to animal [ 42 , 43 ]. Thus, negative results and quantitative differences in defects in response to RNAi against different genes are not interpretable, as it is experimentally challenging to demonstrate quantitative knockdown in the same worm that is scored for subcellular defects and economically not feasible on the scale of the work reported here.…”

“…RNAi using bacterial clones grown as described [ 6 ] was performed with both chronic and acute RNAi experiments as described previously [ 12 ]. A detailed technical description of the strengths, limitations, and caveats to interpretation of results from this screening methodology is available elsewhere [ 43 ].…”