Abstract
BackgroundGenome-scale RNA-interference (RNAi) screens are becoming ever more common gene discovery tools. However, whilst every screen identifies interacting genes, less attention has been given to how factors such as library design and post-screening bioinformatics may be effecting the data generated.ResultsHere we present a new genome-wide RNAi screen of the Drosophila JAK/STAT signalling pathway undertaken in the Sheffield RNAi Screening Facility (SRSF). This screen was carried out using a second-generation, computationally optimised dsRNA library and analysed using current methods and bioinformatic tools. To examine advances in RNAi screening technology, we compare this screen to a biologically very similar screen undertaken in 2005 with a first-generation library. Both screens used the same cell line, reporters and experimental design, with the SRSF screen identifying 42 putative regulators of JAK/STAT signalling, 22 of which verified in a secondary screen and 16 verified with an independent probe design. Following reanalysis of the original screen data, comparisons of the two gene lists allows us to make estimates of false discovery rates in the SRSF data and to conduct an assessment of off-target effects (OTEs) associated with both libraries. We discuss the differences and similarities between the resulting data sets and examine the relative improvements in gene discovery protocols.ConclusionsOur work represents one of the first direct comparisons between first- and second-generation libraries and shows that modern library designs together with methodological advances have had a significant influence on genome-scale RNAi screens.
Highlights
Genome-scale RNA-interference (RNAi) screens are becoming ever more common gene discovery tools
The principal differences between the original Heidelberg Fly Array (HFA) and the repeated Sheffield RNAi Screening Facility (SRSF) screen relate to the libraries used, and whilst the most obvious difference is to the sequences of the Double stranded RNA (dsRNA) that make up the library itself, other factors may be significant
For both HFA and SRSF screens, replicates were considered to be biologically independent of one another with a new batch of transfected cells used for each copy of the genome
Summary
Genome-scale RNA-interference (RNAi) screens are becoming ever more common gene discovery tools. Failure to identify genes that regulate the process of interest represents ‘lost’ information, which is not available for future analysis The frequency of such false negatives can be reduced by improved reagent design and using multiple, independent RNAi reagents per gene [6,7]. The second challenge is the mistaken identification of genes - false positives that incorrectly appear to interact due to edge effects, liquid handling errors or the non-specificity of reagents (known as off-target effects (OTEs); [8,9]) Such false positives can make up more than 50% of primary screen data [6], are likely to confound initial analysis and can only be fully eliminated by downstream secondary screening and gene analysis in vivo
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