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Allele-specific detection of single mRNA molecules in situ


We describe a method for fluorescence in situ identification of individual mRNA molecules, allowing quantitative and accurate measurement, in single cells, of allele-specific transcripts that differ by only a few nucleotides. By using a combination of allele-specific and non–allele-specific probe libraries, we achieve >95% detection accuracy. We investigate the allele-specific stochastic expression of Nanog, which encodes a pluripotency factor, in murine embryonic stem cells.

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Figure 1: Allele-specific in situ detection of single mRNA molecules using SNP-specific probes.
Figure 2: Accurate allele-specific detection using identification probes.


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We thank S. Semrau for programming assistance and B. Panning (University of California, San Francisco) for providing the 2-1 embryonic stem cells and helpful discussions and for inspiring the initial idea that led to the development of this method. We thank J.P. Junker, S. Klemm, Y. Lin, D. Mooijman, A. Pawlosky and Y. Zheng for technical assistance, general discussions and/or comments on the manuscript. This work was supported by a grant (U54CA143874) from the US National Institutes of Health (NIH) National Cancer Institute Physical Sciences Oncology Center at Massachusetts Institute of Technology, an NIH Pioneer Award (8 DP1 CA174420-05) and a Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) Vici award to A.v.O.

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C.H.H. and A.v.O. conceived the method. C.H.H. performed experiments, analyzed the data and wrote the manuscript. A.v.O. guided experiments and data analysis and wrote the manuscript.

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Correspondence to Alexander van Oudenaarden.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–18, Supplementary Table 2 and Supplementary Note (PDF 1768 kb)

Supplementary Table 1

Probe Sequences (TXT 27 kb)

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Hansen, C., van Oudenaarden, A. Allele-specific detection of single mRNA molecules in situ. Nat Methods 10, 869–871 (2013).

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