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Methods Mol Biol. 2016;1478:95-116.

Methods for High-Throughput RNAi Screening in Drosophila Cells.

Author information

1
Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Heidelberg University, Im Neuenheimer Feld 580, D-69120, Heidelberg, Germany.
2
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.

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.

KEYWORDS:

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

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