Methods for high-throughput RNAi screening in drosophila cells

Maximilian Billmann, Michael Boutros

Research output: Chapter in Book/Report/Conference proceedingChapter

6 Scopus citations

Abstract

RNA interference (RNAi) is a potent tool for perturbation of gene function in model organisms and human cells. In Drosophila, efficient RNAi enables screening approaches for components of cellular processes in vivo and in vitro. In cultured cells, measuring the effect of depleting gene products on a genome-wide scale can systematically associate gene function with diverse processes, such as cell growth and proliferation, signaling and trafficking. Here, we describe methods for RNAi experiments in cultured Drosophila cells with a focus on genome-wide loss-of-function screening. We illustrate the design of long double-stranded RNAs and provide protocols for their production by in vitro transcription and delivery in cell-based assays. Furthermore, we provide methods to fine-tune signaling reporters and high-content microscopy assays for genome-wide screening. Finally, we describe essential steps of high-throughput data analysis and how the experimental setup can improve data normalization using a genome-wide RNAi screen for Wnt pathway activity data as an example.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages95-116
Number of pages22
DOIs
StatePublished - Jan 1 2016
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume1478
ISSN (Print)1064-3745

Keywords

  • Cell-based assays
  • Data analysis
  • Double-stranded RNA
  • Drosophila cells
  • High-throughput screening
  • Phenotypic readouts
  • RNAi

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