Abstract
BackgroundHigh-throughput screening using RNAi is a powerful gene discovery method but is often complicated by false positive and false negative results. Whereas false positive results associated with RNAi reagents has been a matter of extensive study, the issue of false negatives has received less attention.ResultsWe performed a meta-analysis of several genome-wide, cell-based Drosophila RNAi screens, together with a more focused RNAi screen, and conclude that the rate of false negative results is at least 8%. Further, we demonstrate how knowledge of the cell transcriptome can be used to resolve ambiguous results and how the number of false negative results can be reduced by using multiple, independently-tested RNAi reagents per gene.ConclusionsRNAi reagents that target the same gene do not always yield consistent results due to false positives and weak or ineffective reagents. False positive results can be partially minimized by filtering with transcriptome data. RNAi libraries with multiple reagents per gene also reduce false positive and false negative outcomes when inconsistent results are disambiguated carefully.
Highlights
High-throughput screening using RNAi is a powerful gene discovery method but is often complicated by false positive and false negative results
We find that the presence of multiple RNAi reagents per gene in a screening library can be a statistically powerful means of reducing false positive and negative results, careful consideration must be made regarding the disambiguation of inconsistent results obtained with multiple reagents directed against the same target gene
Because of the broad functionality of the ribosome and proteasome in basic cell metabolism, we reasoned that double-stranded RNAs (dsRNAs) targeting components of these complexes might affect the output of a wide range of RNAi screens
Summary
High-throughput screening using RNAi is a powerful gene discovery method but is often complicated by false positive and false negative results. The success of RNAi high throughput screening (HTS) relies on low experimental rates of false negative and false positive results, which in turn depend on the efficacy and specificity of the RNAi reagents, respectively (reviewed in [1,2]). False positive results can arise from at least the following causes: experimental noise inherent to large-scale studies, bias associated with a particular screen assay, incorrect gene models, and arguably most importantly, reagent-specific off-target effects (OTEs) (reviewed in [3]). The issue of false positive results associated with RNAi reagents has been a matter of extensive study in recent years for screens in both Drosophila and mammalian cells [6,7,8,9,10,11]. These analyses have suggested that false negative rates may be in the order of 16% to 50% in RNAi HTS
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