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Nat Protoc. 2012 Feb 2;7(2):408-19. doi: 10.1038/nprot.2011.451.

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

Author information

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

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.

PMID:
22301778
PMCID:
PMC4112553
DOI:
10.1038/nprot.2011.451
[Indexed for MEDLINE]
Free PMC Article

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