Abstract
Increasing knowledge about the heterogeneity of mRNA expression within cell populations highlights the need to study transcripts at the level of single cells. We present a method for detection and genotyping of individual transcripts based on padlock probes and in situ target-primed rolling-circle amplification. We detect a somatic point mutation, differentiate between members of a gene family and perform multiplex detection of transcripts in human and mouse cells and tissue.
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Change history
30 July 2010
In the version of this article initially posted online, primer information for detection of mouse α- and β-actin in tissue was incorrect in Online Methods. The error has been corrected in the HTML and PDF versions of the article.
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Acknowledgements
We thank J. Botling at the Department of Genetics and Pathology, Uppsala University for selection of the HER2-positive breast tissue from the Uppsala Biobank, K. Kullander and A. Enjin at the Department of Neuroscience, Uppsala University for providing the mouse embryonic tissue and aiding in tissue identification, C. Wählby at the Department of Genetics and Pathology, Uppsala University for providing MatLab scripts for quantification, L. Lindbom for technical aid, J. Göransson and R. Ke for suggesting formamide in the Ampligase protocol and U. Landegren, S. Fredriksson and T. Sjöblom for comments on the manuscript. Funding was provided by the Knut and Alice Wallenberg Foundation, the Swedish Cancer Foundation, the Göran Gustafsson Foundation, the Erik K. Fernström Foundation, the Swedish Research Council, the European Union FP6 project COMICS and the European Union FP7 (FP7/2007-2013) project READNA (grant agreement HEALTH-F4-2008-201418).
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C.L. and I.G. designed and performed experiments and analyzed data. M.N. supervised. All authors contributed to conceiving the project and writing the manuscript.
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M.N. owns shares in the company Olink AB, Uppsala, Sweden, which holds patents whose value may be affected by publication.
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Larsson, C., Grundberg, I., Söderberg, O. et al. In situ detection and genotyping of individual mRNA molecules. Nat Methods 7, 395–397 (2010). https://doi.org/10.1038/nmeth.1448
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DOI: https://doi.org/10.1038/nmeth.1448
