title: Methods for High-Throughput RNAi Screening in Drosophila Cells.
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 set-up can improve data normalization using a genome-wide RNAi screen for Wnt pathway activity data as an example.
authors.affiliation: Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Heidelberg University, Im Neuenheimer Feld 580, D-69120, Heidelberg, Germany.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Billmann
authors.affiliation: Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Heidelberg University, Im Neuenheimer Feld 580, D-69120, Heidelberg, Germany. m.boutros@dkfz.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Boutros
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D014158:Transcription, Genetic; D051153:Wnt Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D012330:RNA, Double-Stranded; D051153:Wnt Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases
keywords: Cell-based assays; Data analysis; Double-stranded RNA; Drosophila cells; High-throughput screening; Phenotypic readouts; RNAi