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Design and implementation of high-throughput RNAi screens in cultured Drosophila cells

Nature Protocols volume 2, pages 2245–2264 (2007)Cite this article

Abstract

This protocol describes the various steps and considerations involved in planning and carrying out RNA interference (RNAi) genome-wide screens in cultured Drosophila cells. We focus largely on the procedures that have been modified as a result of our experience over the past 3 years and of our better understanding of the underlying technology. Specifically, our protocol offers a set of suggestions and considerations for screen optimization and a step-by-step description of the procedures successfully used at the Drosophila RNAi Screening Center for screen implementation, data collection and analysis to identify potential hits. In addition, this protocol briefly covers postscreen analysis approaches that are often needed to finalize the hit list. Depending on the scope of the screen and subsequent analysis and validation involved, the full protocol can take anywhere from 3 months to 2 years to complete.

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Figure 1: General outline of a genome-wide RNA interference (RNAi) screen.
Figure 2: Comparison of one-step and two-step PCR.
Figure 3: Common approaches for assay detection in RNA interference (RNAi) screens.
Figure 4: Postscreen strategy to evaluate initials hits in a screen.
Figure 5: Examples of acceptable and unacceptable double-stranded RNA (dsRNA) products.
Figure 6: The Heatmap tool: Heatmap views of plates showing experimental artifacts.
Figure 7: Quantile–Quantile plots.
Figure 8: The Heatmap tool: comparison of two normalization methods.

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Acknowledgements

We thank all members of the Perrimon laboratory as well as all past and present DRSC screeners for sharing their experience with us. None of the protocols described here would have been possible without their contributions. Work at the DRSC is supported by grant R01 GM067761 from the NIGMS. N.P. is an investigator of the Howard Hughes Medical School.

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Authors and Affiliations

  1. Department of Genetics and Drosophila RNAi Screening Center, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, 02115, Massachusetts, USA

    Nadire Ramadan, Ian Flockhart, Matthew Booker, Norbert Perrimon & Bernard Mathey-Prevot

  2. Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, 02115, Massachusetts, USA

    Norbert Perrimon

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Correspondence to Bernard Mathey-Prevot.

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Ramadan, N., Flockhart, I., Booker, M. et al. Design and implementation of high-throughput RNAi screens in cultured Drosophila cells. Nat Protoc 2, 2245–2264 (2007). https://doi.org/10.1038/nprot.2007.250

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