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

Nature Protocols volume 7, pages 408–419 (2012)Cite this article

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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.

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

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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.)

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Authors and Affiliations

  1. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, USA

    Tatjana Trcek, Jeffrey A Chao, Hye Yoon Park, Shailesh M Shenoy & Robert H Singer

  2. National Cancer Institute, US National Institutes of Health, Bethesda, Maryland, USA

    Daniel R Larson

  3. Département de Biochimie, Université de Montréal, Montréal, Québec, Canada

    Daniel Zenklusen

  4. Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA

    Robert H Singer

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  1. Tatjana Trcek
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  6. Shailesh M Shenoy
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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.

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Correspondence to Robert H Singer.

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Competing interests

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

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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

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