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

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Drosophila

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1478))

Abstract

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.

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Acknowledgements

We thank Reinhard Fässler for generous support, Bianca Habermann for help with the bioinformatics section, and Maria Spletter and the entire Schnorrer lab for informative suggestions on this manuscript. Our laboratory is supported by the Max Planck Society, Humboldt Foundation and EMBO postdoctoral fellowships (A.K.-Ç.), the EMBO Young Investigator Program (F.S.) and the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant 310939.

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  1. Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152, Martinsried, Germany

    Aynur Kaya-Çopur & Frank Schnorrer

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  1. Aynur Kaya-Çopur
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Correspondence to Frank Schnorrer .

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  1. Institute of Genetics, Technische Universität Dresden, Dresden, Germany

    Christian Dahmann

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Kaya-Çopur, A., Schnorrer, F. (2016). A Guide to Genome-Wide In Vivo RNAi Applications in Drosophila . In: Dahmann, C. (eds) Drosophila. Methods in Molecular Biology, vol 1478. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6371-3_6

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  • DOI: https://doi.org/10.1007/978-1-4939-6371-3_6

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