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Combined RNAi and localization for functionally dissecting long noncoding RNAs

Nature Methods volume 9, pages 360–362 (2012)Cite this article

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Abstract

Whereas methods to comprehensively study cellular roles of protein-coding genes are available, techniques to systematically investigate long noncoding RNAs (lncRNAs), which have been implicated in diverse biological pathways, are limited. Here we report combined knockdown and localization analysis of noncoding RNAs (c-KLAN) that merges functional characterization and localization approaches to study lncRNAs. Using this technique we identified transcripts that regulate mouse embryonic stem cell identity.

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Figure 1: c-KLAN and its applications.
Figure 2: Implementation of c-KLAN to study the role of Panct1 in mouse ESCs.

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Acknowledgements

We thank S. Rose, A.Weise and R. Zschau for help in esiRNA library synthesis, I. De Vries and I. Nusslein for technical assistance, D. White and J. Schindelin for help with Deltavision imaging and postprocessing, A. Smith (University of Cambridge) for the Oct4-Gip cell line and all members of the Buchholz laboratory for helpful discussions and suggestions. This work was supported by the Bundesministerium für Bildung und Forschung grant GO-Bio (0315980), EU FP7 grant SyBoSS 242129, and the Deutsche Forschungsgemeinschaft grants SPP1356 BU1400/3-1 and SFB655.

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Authors and Affiliations

  1. Department of Medical Systems Biology, University of Technology Dresden, University Hospital and Medical Faculty Carl Gustav Carus, Dresden, Germany

    Debojyoti Chakraborty, Dennis Kappei, Mirko Theis, Anja Nitzsche, Li Ding, Maciej Paszkowski-Rogacz, Nicolas Berger & Frank Buchholz

  2. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

    Debojyoti Chakraborty, Dennis Kappei, Mirko Theis, Anja Nitzsche, Li Ding, Maciej Paszkowski-Rogacz, Vineeth Surendranath, Nicolas Berger & Frank Buchholz

  3. Eupheria Biotech GmbH, Dresden, Germany

    Mirko Theis

  4. Max Delbruck Center of Molecular Medicine, Berlin, Germany

    Herbert Schulz, Kathrin Saar & Norbert Hubner

Authors
  1. Debojyoti Chakraborty
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  2. Dennis Kappei
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  12. Frank Buchholz
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Contributions

D.C., F.B., D.K. and M.T. conceived and designed experiments. D.C. performed the screen, RNA FISH and other major experiments. M.T. and D.K. contributed to the synthesis of esiRNA library against lncRNAs. A.N. performed ESC differentiation experiments. M.P.-R. analyzed microarray data. V.S. performed in silico analysis for library production. N.B. contributed to knockdown comparison experiments. L.D., H.S., K.S. and N.H. did microarray experiments. D.C. and F.B. prepared the manuscript.

Corresponding author

Correspondence to Frank Buchholz.

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Competing interests

M.T. is a staff member of Eupheria Biotech GmbH. F.B. is an advisory member of Eupheria Biotech GmbH.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Tables 3–6 and Supplementary Notes (PDF 7616 kb)

Supplementary Table 1

Complete list of lncRNAs with their accession numbers and DEQOR optimized primer sequences used for library synthesis. (XLSX 133 kb)

Supplementary Table 2

List of 594 esiRNAs against lncRNAs that were successfully synthesized by c-KLAN approach. (XLSX 41 kb)

Supplementary Table 7

Primers used in this study. (XLSX 13 kb)

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Chakraborty, D., Kappei, D., Theis, M. et al. Combined RNAi and localization for functionally dissecting long noncoding RNAs. Nat Methods 9, 360–362 (2012). https://doi.org/10.1038/nmeth.1894

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