@article {46725,
	title = {Single-mRNA counting using fluorescent in situ hybridization in budding yeast.},
	journal = {Nature Protocols},
	volume = {7},
	year = {2012},
	month = {2012 Feb 2},
	pages = {408-19},
	abstract = {<p>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.</p>
},
	keywords = {Algorithms, Antisense Elements (Genetics), In Situ Hybridization, Fluorescence, RNA, Fungal, RNA, Messenger, Saccharomyces cerevisiae},
	issn = {1750-2799},
	doi = {10.1038/nprot.2011.451},
	author = {Trcek, Tatjana and Chao, Jeffrey A and Larson, Daniel R and Park, Hye Yoon and Zenklusen, Daniel and Shenoy, Shailesh M and Singer, Robert H}
}
