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In situ sequencing for RNA analysis in preserved tissue and cells


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.


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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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Authors and Affiliations



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.

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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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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).

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