A Guide to Genome-Wide In Vivo RNAi Applications in Drosophila

Methods Mol Biol. 2016;1478:117-143. doi: 10.1007/978-1-4939-6371-3_6.


RNAi technologies enable the testing of gene function in a cell-type- and stage-specific manner in Drosophila. The development of genome-wide RNAi libraries has allowed expansion of this approach to the genome scale and supports identification of most genes required for a given process in a cell type of choice. However, a large-scale RNAi approach also harbors many potential pitfalls that can complicate interpretation of the results. Here, we summarize published screens and provide a guide on how to optimally plan and perform a large-scale, in vivo RNAi screen. We highlight the importance of assay design and give suggestions on how to optimize the assay conditions by testing positive and negative control genes. These genes are used to estimate false-negative and false-positive rates of the screen data. We discuss the planning and logistics of a large-scale screen in detail and suggest bioinformatics platforms to identify and select gene groups of interest for secondary assays. Finally, we review various options to confirm RNAi knock-down specificity and thus identify high confidence genes for more detailed case-by-case studies in the future.

Keywords: Dicer-2; Drosophila; False-discovery; False-negative; False-positive; GAL4-UAS; Genetic screens; High-throughput screens; In vivo; NIG-FLY; Off-target effects; RNAi; TRiP; VDRC.

Publication types

  • Review
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Computational Biology / methods
  • Databases, Genetic
  • Drosophila Proteins / genetics*
  • Drosophila Proteins / metabolism
  • Drosophila melanogaster / genetics*
  • Drosophila melanogaster / metabolism
  • Gene Expression Profiling
  • Genetic Techniques
  • Genome*
  • Genomic Library
  • High-Throughput Screening Assays*
  • RNA Helicases / genetics*
  • RNA Helicases / metabolism
  • RNA Interference*
  • RNA, Double-Stranded / genetics
  • RNA, Double-Stranded / metabolism
  • Ribonuclease III / genetics*
  • Ribonuclease III / metabolism
  • Signal Transduction
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism
  • Transcription, Genetic


  • Drosophila Proteins
  • GAL4 protein, Drosophila
  • RNA, Double-Stranded
  • Transcription Factors
  • DCR-2 protein, Drosophila
  • Ribonuclease III
  • RNA Helicases