Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Single-mRNA counting using fluorescent in situ hybridization in budding yeast

Abstract

Fluorescent in situ hybridization (FISH) allows the quantification of single mRNAs in budding yeast using fluorescently labeled single-stranded DNA probes, a wide-field epifluorescence microscope and a spot-detection algorithm. Fixed yeast cells are attached to coverslips and hybridized with a mixture of FISH probes, each conjugated to several fluorescent dyes. Images of cells are acquired in 3D and maximally projected for single-molecule analysis. Diffraction-limited labeled mRNAs are observed as bright fluorescent spots and can be quantified using a spot-detection algorithm. FISH preserves the spatial distribution of cellular RNA distribution within the cell and the stochastic fluctuations in individual cells that can lead to phenotypic differences within a clonal population. This information, however, is lost if the RNA content is measured on a population of cells by using reverse transcriptase PCR, microarrays or high-throughput sequencing. The FISH procedure and image acquisition described here can be completed in 3 d.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Single-mRNA detection and counting.
Figure 2: Single-molecule FISH controls.

References

  1. Singer, R.H. & Ward, D.C. Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog. Proc. Natl. Acad. Sci. USA 79, 7331–7335 (1982).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Lawrence, J.B. & Singer, R.H. Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucleic Acids Res. 13, 1777–1799 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  4. Volpi, E.V. & Bridger, J.M. FISH glossary: an overview of the fluorescence in situ hybridization technique. Biotechniques 45, 385–386, 388, 390 passim (2008).

    Article  CAS  PubMed  Google Scholar 

  5. Lawrence, J.B. & Singer, R.H. Intracellular localization of messenger RNAs for cytoskeletal proteins. Cell 45, 407–415 (1986).

    Article  CAS  PubMed  Google Scholar 

  6. Long, R.M. et al. Mating type switching in yeast controlled by asymmetric localization of ASH1 mRNA. Science 277, 383–387 (1997).

    Article  CAS  PubMed  Google Scholar 

  7. Gall, J.G. & Pardue, M.L. Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc. Natl. Acad. Sci. USA 63, 378–383 (1969).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kislauskis, E.H. et al. Isoform-specific 3′-untranslated sequences sort α-cardiac and β-cytoplasmic actin messenger RNAs to different cytoplasmic compartments. J. Cell Biol. 123, 165–172 (1993).

    Article  CAS  PubMed  Google Scholar 

  9. Femino, A.M. et al. Visualization of single RNA transcripts in situ. Science 280, 585–590 (1998).

    Article  CAS  PubMed  Google Scholar 

  10. Levsky, J.M. et al. Single-cell gene expression profiling. Science 297, 836–840 (2002).

    Article  CAS  PubMed  Google Scholar 

  11. Gandhi, S.J. et al. Transcription of functionally related constitutive genes is not coordinated. Nat. Struct. Mol. Biol. 18, 27–34 (2011).

    Article  CAS  PubMed  Google Scholar 

  12. Zenklusen, D., Larson, D.R. & Singer, R.H. Single-RNA counting reveals alternative modes of gene expression in yeast. Nat. Struct. Mol. Biol. 15, 1263–1271 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Silverman, S.J. et al. Metabolic cycling in single yeast cells from unsynchronized steady-state populations limited on glucose or phosphate. Proc. Natl. Acad. Sci. USA 107, 6946–6951 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Garcia, M. et al. Mitochondria-associated yeast mRNAs and the biogenesis of molecular complexes. Mol. Biol. Cell 18, 362–368 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Trcek, T. et al. Single-molecule mRNA decay measurements reveal promoter-regulated mRNA stability in yeast. Cell 147, 1484–1497 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  Google Scholar 

  17. Thompson, R.E., Larson, D.R. & Webb, W.W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Femino, A.M. et al. Visualization of single molecules of mRNA in situ. Methods Enzymol. 361, 245–304 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Larson, D.R. et al. Visualization of retrovirus budding with correlated light and electron microscopy. Proc. Natl. Acad. Sci. USA 102, 15453–15458 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Raj, A. et al. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Raj, A. & Tyagi, S. Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes. Methods Enzymol. 472, 365–386 (2010).

    Article  CAS  PubMed  Google Scholar 

  22. Youk, H., Raj, A. & van Oudenaarden, A. Imaging single mRNA molecules in yeast. Methods Enzymol. 470, 429–446 (2010).

    Article  CAS  PubMed  Google Scholar 

  23. Bertrand, E. et al. Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445 (1998).

    Article  CAS  PubMed  Google Scholar 

  24. Park, H.Y., Buxbaum, A.R. & Singer, R.H. Single mRNA tracking in live cells. Methods Enzymol. 472, 387–406 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Tyagi, S. Imaging intracellular RNA distribution and dynamics in living cells. Nat. Methods 6, 331–338 (2009).

    Article  CAS  PubMed  Google Scholar 

  26. Chao, J.A. et al. Structural basis for the coevolution of a viral RNA-protein complex. Nat. Struct. Mol. Biol. 15, 103–105 (2008).

    Article  CAS  PubMed  Google Scholar 

  27. Larson, D.R. et al. Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science 332, 475–478 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Daigle, N. & Ellenberg, J. λN-GFP: an RNA reporter system for live-cell imaging. Nat. Methods 4, 633–636 (2007).

    Article  CAS  PubMed  Google Scholar 

  29. Takizawa, P.A. & Vale, R.D. The myosin motor, Myo4p, binds Ash1 mRNA via the adapter protein, She3p. Proc. Natl. Acad. Sci. USA 97, 5273–5278 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lange, S. et al. Simultaneous transport of different localized mRNA species revealed by live-cell imaging. Traffic 9, 1256–1267 (2008).

    Article  CAS  PubMed  Google Scholar 

  31. Zenklusen, D. & Singer, R.H. Analyzing mRNA expression using single mRNA resolution fluorescent in situ hybridization. Methods Enzymol. 470, 641–659 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Spellman, P.T. et al. Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Mol. Biol. Cell 9, 3273–3297 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Long, R.M. et al. She2p is a novel RNA-binding protein that recruits the Myo4p-She3p complex to ASH1 mRNA. EMBO J. 19, 6592–6601 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the Singer laboratory members for critical review of this manuscript. This work was supported by US National Institutes of Health grants GM57071 and GM86217 (awarded to R.H.S.) and a grant from the Canadian Institutes of Health Research (awarded to D.Z.)

Author information

Authors and Affiliations

Authors

Contributions

T.T. designed and generated the figures in the paper. T.T., J.A.C., D.Z. contributed to optimization of the multiprobe FISH protocol. D.R.L. wrote the detection algorithm Localize. S.M.S. wrote the cell segmentation plug-in for ImageJ. T.T., J.A.C., D.R.L., H.Y.P., D.Z. and R.H.S. wrote the paper.

Corresponding author

Correspondence to Robert H Singer.

Ethics declarations

Competing interests

R.H.S. is a consultant with Biosearch Technologies.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Trcek, T., Chao, J., Larson, D. et al. Single-mRNA counting using fluorescent in situ hybridization in budding yeast. Nat Protoc 7, 408–419 (2012). https://doi.org/10.1038/nprot.2011.451

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.451

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing