False negative rates in Drosophila cell-based RNAi screens: a case study

Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
BMC Genomics (Impact Factor: 4.04). 01/2011; 12:50. DOI: 10.1186/1471-2164-12-50
Source: PubMed

ABSTRACT High-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.
We 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.
RNAi 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.

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Available from: Ian Flockhart, Sep 02, 2015
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    • "While many genes and candidate-loci involved in the specification or maintenance dendrite morphology have been identified using forward genetic, gain-of-function and RNAi screens [8]–[14], we remain far from having a coherent mechanistic understanding of the processes governing class-specific dendrite development. Further, RNAi screens, without being guided by cell-type specific transcriptomic information, have frequently been observed to result in high false positive rates and ambiguous results [15]. In addition, many genes that contribute to complex morphogenesis programs may function in a range of developmental processes and are thereby expected to exhibit pleiotropy which can result in a failure to identify such morphogenesis genes in standard genetic screens [16]. "
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