High-throughput RNAi screening by time-lapse imaging of live human cells (original) (raw)

Nature Methods volume 3, pages 385–390 (2006)Cite this article

Abstract

RNA interference (RNAi) is a powerful tool to study gene function in cultured cells. Transfected cell microarrays in principle allow high-throughput phenotypic analysis after gene knockdown by microscopy. But bottlenecks in imaging and data analysis have limited such high-content screens to endpoint assays in fixed cells and determination of global parameters such as viability. Here we have overcome these limitations and developed an automated platform for high-content RNAi screening by time-lapse fluorescence microscopy of live HeLa cells expressing histone-GFP to report on chromosome segregation and structure. We automated all steps, including printing transfection-ready small interfering RNA (siRNA) microarrays, fluorescence imaging and computational phenotyping of digital images, in a high-throughput workflow. We validated this method in a pilot screen assaying cell division and delivered a sensitive, time-resolved phenoprint for each of the 49 endogenous genes we suppressed. This modular platform is scalable and makes the power of time-lapse microscopy available for genome-wide RNAi screens.

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Acknowledgements

We thank S. Narumiya (Kyoto University, Kyoto) and T. Hirota (Institute of Molecular Pathology; IMP; Vienna) for HeLa 'Kyoto' cells; W. Huber (European Bioinformatics Institute; EBI; Hinxton) for advice on statistical analysis of kinetic data; O. Gruss (Zentrum für Molekulare Biologie Heidelberg; ZNBH; Heidelberg) for TPX2 antibody; J.-M. Peters (IMP; Vienna) for RPE cells; I. Hoffmann (Deutsches Krebsforschungszentrum; DKFZ; Heidelberg) for U2OS cells; H. Runz (Univ. Heidelberg) for primary human fibroblasts; Chroma Inc. for providing customized emission filter sets free of charge; EMBL's IT Services group (B. Kindler, M. Hemberger, R. Lück) for support; Olympus Biosystems, Hamilton and Bio-Rad for continuous support; Cenix BioScience GmbH for siRNA design and for providing the A549 cells; and Ambion Europe, Ltd. for providing siRNAs for validation. This project was funded by grants to J.E. within the MitoCheck consortium by the European Commission (FP6-503464) as well as in part by the Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network (NGFN) (NGFN-2 SMP-RNAi, FKZ01GR0403 to J.E. and NGFN-2 SMP-Cell FKZ01GR0423, NGFN-1 FKZ01GR0101, FKZ01KW0013 to R.P.).

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Author notes

  1. Beate Neumann, Michael Held, Urban Liebel and Holger Erfle: These authors contributed equally to this work.

Authors and Affiliations

  1. MitoCheck Project Group, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, D-69117, Germany
    Beate Neumann, Michael Held, Urban Liebel, Holger Erfle & Phill Rogers
  2. Cell Biology/Biophysics, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, D-69117, Germany
    Rainer Pepperkok & Jan Ellenberg
  3. Gene Expression Programmes, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, D-69117, Germany
    Jan Ellenberg

Authors

  1. Beate Neumann
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  2. Michael Held
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  3. Urban Liebel
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  4. Holger Erfle
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  5. Phill Rogers
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  6. Rainer Pepperkok
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  7. Jan Ellenberg
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Corresponding author

Correspondence toJan Ellenberg.

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The authors declare no competing financial interests.

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Neumann, B., Held, M., Liebel, U. et al. High-throughput RNAi screening by time-lapse imaging of live human cells.Nat Methods 3, 385–390 (2006). https://doi.org/10.1038/nmeth876

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