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Single-molecule mRNA detection and counting in mammalian tissue


We present a protocol for visualizing and quantifying single mRNA molecules in mammalian (mouse and human) tissues. In the approach described here, sets of about 50 short oligonucleotides, each labeled with a single fluorophore, are hybridized to target mRNAs in tissue sections. Each set binds to a single mRNA molecule and can be detected by fluorescence microscopy as a diffraction-limited spot. Tissue architecture is then assessed by counterstaining the sections with DNA dye (DAPI), and cell borders can be visualized with a dye-coupled antibody. Spots are detected automatically with custom-made software, which we make freely available. The mRNA molecules thus detected are assigned to single cells within a tissue semiautomatically by using a graphical user interface developed in our laboratory. In this protocol, we describe an example of quantitative analysis of mRNA levels and localization in mouse small intestine. The procedure (from tissue dissection to obtaining data sets) takes 3 d. Data analysis will require an additional 3–7 d, depending on the type of analysis.

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Figure 1: Single-molecule RNA detection and quantification in mouse small intestinal cryosections.
Figure 2: Screenshot of the workspace in the image analysis GUI ImageM, taken during cell segmentation.
Figure 3: Screenshot of the ImageM workspace, taken during the process of automated detection of diffraction-limited spots.
Figure 4: Hybridization setup.
Figure 5: Positive and negative controls for smFISH.


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Authors and Affiliations



A.v.O. supervised the project. A.L., S.I. and J.P.J. developed the protocol. X.W. and Z.P.F. contributed to software development. A.L., S.I., J.P.J. and A.v.O. wrote the paper.

Corresponding author

Correspondence to Alexander van Oudenaarden.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Sequences of the target mRNAs and probe oligonucleotide probe libraries used in the example experiment. (PDF 162 kb)

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Lyubimova, A., Itzkovitz, S., Junker, J. et al. Single-molecule mRNA detection and counting in mammalian tissue. Nat Protoc 8, 1743–1758 (2013).

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