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

Tatjana Trcek, Jeffrey A. Chao, Daniel R. Larson, Hye Yoon Park, Daniel Zenklusen, Shailesh M. Shenoy, Robert H. Singer

Research output: Contribution to journalArticlepeer-review

75 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)408-419
Number of pages12
JournalNature Protocols
Volume7
Issue number2
DOIs
StatePublished - Feb 2012
Externally publishedYes

Bibliographical note

Funding Information:
acknowleDGMents 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 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.

Fingerprint

Dive into the research topics of 'Single-mRNA counting using fluorescent in situ hybridization in budding yeast'. Together they form a unique fingerprint.

Cite this