title: Advances in genome-wide RNAi cellular screens: a case study using the Drosophila JAK/STAT pathway.
abstract: BACKGROUND
  Genome-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.
  
  
  RESULTS
  Here 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.
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: The MRC Centre for Developmental and Biomedical Genetics and The Department of Biomedical Science, University of Sheffield, Firth Court,Western Bank, Sheffield S10 2TN, UK.
authors.forename: Katherine H
authors.initials: KH
authors.lastname: Fisher
authors.forename: Victoria M
authors.initials: VM
authors.lastname: Wright
authors.forename: Amy
authors.initials: A
authors.lastname: Taylor
authors.forename: Martin P
authors.initials: MP
authors.lastname: Zeidler
authors.forename: Stephen
authors.initials: S
authors.lastname: Brown
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics*; D005786:Gene Expression Regulation; D015723:Gene Library; D017930:Genes, Reporter; D049750:Genome, Insect; D055106:Genome-Wide Association Study; D053612:Janus Kinases / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases; D010957:Plasmids; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D050791:STAT Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D029721:Drosophila Proteins; D034741:RNA, Small Interfering; D050791:STAT Transcription Factors; D014157:Transcription Factors; D008156:Luciferases; D053612:Janus Kinases; C085845:hop protein, Drosophila