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Single-molecule mRNA detection and counting in mammalian tissue

Nature Protocols volume 8, pages 1743–1758 (2013)Cite this article

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Abstract

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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References

  1. Femino, A.M., Fay, F.S., Fogarty, K. & Singer, R.H. Visualization of single RNA Transcripts in situ. Science 280, 585–590 (1998).

    Article  CAS  Google Scholar 

  2. Raj, A., van den Bogaard, P., Rifkin, S.A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

    Article  CAS  Google Scholar 

  3. Itzkovitz, S. et al. Single-molecule transcript counting of stem-cell markers in the mouse intestine. Nat. Cell Biol. 14, 106–114 (2012).

    Article  CAS  Google Scholar 

  4. Itzkovitz, S., Blat, I.C., Jacks, T., Clevers, H. & van Oudenaarden, A. Optimality in the development of intestinal crypts. Cell 148, 608–619 (2012).

    Article  CAS  Google Scholar 

  5. Muñoz, J. et al. The Lgr5 intestinal stem cell signature: robust expression of proposed quiescent '+4' cell markers. EMBO J. 31, 3079–3091 (2012).

    Article  Google Scholar 

  6. Van Es, J.H. et al. A critical role for the Wnt effector Tcf4 in adult intestinal homeostatic self-renewal. Mol. Cell Biol. 32, 1918–1927 (2012).

    Article  CAS  Google Scholar 

  7. Guo, W. et al. Slug and Sox9 cooperatively determine the mammary stem cell state. Cell 148, 1015–1028 (2012).

    Article  CAS  Google Scholar 

  8. Barker, N. et al. Lgr5+ve stem/progenitor cells contribute to nephron formation during kidney development. Cell Rep. 2, 540–552 (2012).

    Article  CAS  Google Scholar 

  9. Bienko, M. et al. A versatile genome-scale PCR-based pipeline for high-definition DNA FISH. Nat. Methods 10, 122–124 (2013).

    Article  CAS  Google Scholar 

  10. Levesque, M.J. & Raj, A. Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation. Nat. Methods 10, 246–248 (2013).

    Article  CAS  Google Scholar 

  11. Levsky, J.M., Shenoy, S.M., Pezo, R.C. & Singer, R.H. Single-cell gene expression profiling. Science 297, 836–840 (2002).

    Article  CAS  Google Scholar 

  12. Snippert, H.J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).

    Article  CAS  Google Scholar 

  13. Itzkovitz, S. & van Oudenaarden, A. Validating transcripts with probes and imaging technology. Nat. Methods 8, S12–S19 (2011).

    Article  CAS  Google Scholar 

  14. Levsky, J.M. & Singer, R.H. Fluorescence in situ hybridization: past, present and future. J. Cell Sci. 116, 2833–2838 (2003).

    Article  CAS  Google Scholar 

  15. Emmert-Buck, M.R. et al. Laser-capture microdissection. Science 274, 998–1001 (1996).

    Article  CAS  Google Scholar 

  16. Larsson, C., Grundberg, I., Söderberg, O. & Nilsson, M. In situ detection and genotyping of individual mRNA molecules. Nat. Methods 7, 395–397 (2010).

    Article  CAS  Google Scholar 

  17. Morin, R. et al. Profiling the HeLa S3 transcriptome using randomly primed cDNA and massively parallel short-read sequencing. BioTechniques 45, 81–94 (2008).

    Article  CAS  Google Scholar 

  18. Sultan, M. et al. A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome. Science 321, 956–960 (2008).

    Article  CAS  Google Scholar 

  19. Tang, F., Lao, K. & Surani, M.A. Development and applications of single-cell transcriptome analysis. Nat. Methods 8, S6–S11 (2011).

    Article  CAS  Google Scholar 

  20. Lubeck, E. & Cai, L. Single-cell systems biology by super-resolution imaging and combinatorial labeling. Nat. Methods 9, 743–748 (2012).

    Article  CAS  Google Scholar 

  21. Kilkenny, C, Browne, W.J., Cuthill, I.C., Emerson, M. & Altman, D.G. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 8, e1000412 (2010).

    Article  Google Scholar 

  22. Maronpot, R.R., Boorman, G.A. & Gaul, B.W. Pathology of the Mouse: Reference and Atlas (Cache River Press, 1999).

  23. Cook, M.J. The Anatomy of the Laboratory Mouse (Academic Press, 1965).

  24. Carpenter, A.E. et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100 (2006).

    Article  Google Scholar 

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Author information

Author notes

  1. Anna Lyubimova and Shalev Itzkovitz: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physics, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Anna Lyubimova, Shalev Itzkovitz, Jan Philipp Junker & Alexander van Oudenaarden

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Anna Lyubimova, Shalev Itzkovitz, Jan Philipp Junker & Alexander van Oudenaarden

  3. Hubrecht Institute—KNAW (Royal Netherlands Academy of Arts and Sciences) and University Medical Center Utrecht, Utrecht, Netherlands.,

    Anna Lyubimova, Jan Philipp Junker & Alexander van Oudenaarden

  4. Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel

    Shalev Itzkovitz

  5. Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Zi Peng Fan

  6. Computational and Systems Biology Graduate Program, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Zi Peng Fan & Xuebing Wu

  7. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Xuebing Wu & Alexander van Oudenaarden

Authors
  1. Anna Lyubimova
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  2. Shalev Itzkovitz
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  4. Zi Peng Fan
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  5. Xuebing Wu
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  6. Alexander van Oudenaarden
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Contributions

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). https://doi.org/10.1038/nprot.2013.109

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