Abstract
RNA interference (RNAi) is a technique widely used for gene silencing in organisms and cultured cells, and depends on sequence homology between double-stranded RNA (dsRNA) and target mRNA molecules. Numerous cell-based genome-wide screens have successfully identified novel genes involved in various biological processes, including signal transduction, cell viability/death, and cell morphology. However, cell-based screens cannot address cellular processes such as development, behavior, and immunity. Drosophila and Caenorhabditis elegans are two model organisms whose whole bodies and individual body parts have been subjected to RNAi-based genome-wide screening. Moreover, Drosophila RNAi allows the manipulation of gene function in a spatiotemporal manner when it is implemented using the Gal4/UAS system. Using this inducible RNAi technique, various large-scale screens have been performed in Drosophila, demonstrating that the method is straightforward and valuable. However, accumulated results reveal that the results of RNAi-based screens have relatively high levels of error, such as false positives and negatives. Here, we review in vivo RNAi screens in Drosophila and the methods that could be used to remove ambiguity from screening results.
Highlights
The classical forward genetic approach is a powerful method for the elucidation of genetic and molecular mechanisms
RNA interference (RNAi) screens lead to high numbers of both false positive and false negative results, in some cases this may be due to differences in the approaches used for screening
One potential source of false positives in RNAi-based screens comes from off-target effects (OTEs) that occur when a double-stranded RNA (dsRNA) has homology to messenger RNA (mRNA) that are not the intended target
Summary
The classical forward genetic approach is a powerful method for the elucidation of genetic and molecular mechanisms. RNAs (siRNAs), long double-stranded RNAs (dsRNAs), or short hairpin RNAs (shRNAs) to target mRNA molecules for degradation [6,7,8] This technique has been widely and intensively applied in gene silencing experiments. Complete genome sequences provide information about the organization and transcribed sequences of all genes in a genome Based on this information, dsRNAs can be designed to efficiently and reduce the expression of targeted genes. RNAi has two major problems; the delivery and/or expression of dsRNAs in target cells and erroneous results, such as false positives and negatives. False positives, where genes are unexpectedly silenced by dsRNAs, are caused by off-target effects (OTEs) and indirect effects by knockdown of general machineries, for example, general transcription machinery, whereas false negatives are mainly caused by the low silencing efficacies of specific dsRNA molecules. We focus mainly on organism-based (hereafter referred to as in vivo) RNAi screens and experimental and computational disambiguation methods that could be used for check the results of these screens
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