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In situ sequencing for RNA analysis in preserved tissue and cells

Nature Methods volume 10, pages 857–860 (2013)Cite this article

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Abstract

Tissue gene expression profiling is performed on homogenates or on populations of isolated single cells to resolve molecular states of different cell types. In both approaches, histological context is lost. We have developed an in situ sequencing method for parallel targeted analysis of short RNA fragments in morphologically preserved cells and tissue. We demonstrate in situ sequencing of point mutations and multiplexed gene expression profiling in human breast cancer tissue sections.

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Figure 1: Procedure for targeted in situ sequencing.
Figure 2: In situ sequencing of fragments of ACTB and HER2 mRNA in breast cancer tissue.
Figure 3: Gene expression profiling on an ER-negative breast cancer tissue section by barcode-targeted in situ sequencing.

References

  1. Levsky, J.M. & Singer, R.H. Trends Cell Biol. 13, 4–6 (2003).

    Article  CAS  Google Scholar 

  2. Wang, Z., Gerstein, M. & Snyder, M. Nat. Rev. Genet. 10, 57–63 (2009).

    Article  CAS  Google Scholar 

  3. Bonner, R.F. et al. Science 278, 1481–1483 (1997).

    Article  CAS  Google Scholar 

  4. Dalerba, P. et al. Nat. Biotechnol. 29, 1120–1127 (2011).

    Article  CAS  Google Scholar 

  5. Navin, N. et al. Nature 472, 90–94 (2011).

    Article  CAS  Google Scholar 

  6. Tang, F. et al. Nat. Methods 6, 377–382 (2009).

    Article  CAS  Google Scholar 

  7. Hou, Y. et al. Cell 148, 873–885 (2012).

    Article  CAS  Google Scholar 

  8. Xu, X. et al. Cell 148, 886–895 (2012).

    Article  CAS  Google Scholar 

  9. Nilsson, M. et al. Science 265, 2085–2088 (1994).

    Article  CAS  Google Scholar 

  10. Banér, J., Nilsson, M., Mendel-Hartvig, M. & Landegren, U. Nucleic Acids Res. 26, 5073–5078 (1998).

    Article  Google Scholar 

  11. Larsson, C., Grundberg, I., Soderberg, O. & Nilsson, M. Nat. Methods 7, 395–397 (2010).

    Article  CAS  Google Scholar 

  12. Shendure, J. et al. Science 309, 1728–1732 (2005).

    Article  CAS  Google Scholar 

  13. Drmanac, R. et al. Science 327, 78–81 (2010).

    Article  CAS  Google Scholar 

  14. Kamentsky, L. et al. Bioinformatics 27, 1179–1180 (2011).

    Article  CAS  Google Scholar 

  15. Thévenaz, P., Ruttimann, U.E. & Unser, M. IEEE Trans. Image Process. 7, 27–41 (1998).

    Article  Google Scholar 

  16. Sparano, J.A. & Paik, S. J. Clin. Oncol. 26, 721–728 (2008).

    Article  Google Scholar 

  17. Wang, E.T. et al. Nature 456, 470–476 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank J. Lee, G.M. Church and F. Pontén for valuable discussion about this work. We thank M. Dahlberg for helping extracting RNA-seq data from publications. The research reported in this paper was funded by the Swedish Research Council, VINNOVA project “Companion diagnostic initiative,” the European Community's 7th Framework Program (FP7/2007-2013) under grant agreement nos. 259796 (DiaTools) and 201418 (READNA), the Science for Life Laboratory, Stockholm and Uppsala, and the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115234 (OncoTrack).

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Author notes

  1. Rongqin Ke and Marco Mignardi: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Stockholm, Sweden

    Rongqin Ke, Marco Mignardi, Jessica Svedlund & Mats Nilsson

  2. Department of Immunology, Genetics, and Pathology, the Rudbeck Laboratory, Uppsala University, Uppsala, Sweden

    Rongqin Ke, Marco Mignardi, Johan Botling & Mats Nilsson

  3. Science for Life Laboratory, Centre for Image Analysis, Uppsala University, Uppsala, Sweden

    Alexandra Pacureanu & Carolina Wählby

  4. Imaging Platform, Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Carolina Wählby

Authors
  1. Rongqin Ke
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  2. Marco Mignardi
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  3. Alexandra Pacureanu
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  4. Jessica Svedlund
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  5. Johan Botling
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  6. Carolina Wählby
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  7. Mats Nilsson
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Contributions

R.K. and M.M. designed and performed the experiments. J.B. provided tissue sections and pathology examination of the tissue. C.W. designed the image analysis pipelines and performed the image analysis together with A.P. J.S. performed the correlation between in situ sequencing and RNA-seq data. R.K., M.M., C.W. and M.N. wrote the manuscript. All authors commented on and revised the manuscript. M.N. conceived the idea and supervised the project.

Corresponding authors

Correspondence to Carolina Wählby or Mats Nilsson.

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Competing interests

M.N. owns shares in the company Olink AB, Uppsala, Sweden, which holds patents whose value may be affected by publication of these results.

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Supplementary Figures 1–15, Supplementary Tables 1–7 and Supplementary Notes 1 and 2 (PDF 3726 kb)

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Ke, R., Mignardi, M., Pacureanu, A. et al. In situ sequencing for RNA analysis in preserved tissue and cells. Nat Methods 10, 857–860 (2013). https://doi.org/10.1038/nmeth.2563

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