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Nucleic Acids Res. 2017 Sep 6;45(15):e141. doi: 10.1093/nar/gkx568.

mRNA detection in budding yeast with single fluorophores.

Author information

1
School of Physics, Georgia Institute of Technology, 837 State Street, Atlanta, GA 30332-0430, USA.
2
Department of Biology, The Catholic University of America, 620 Michigan Avenue NE, Washington, DC 20064, USA.

Abstract

Quantitative measurement of mRNA levels in single cells is necessary to understand phenotypic variability within an otherwise isogenic population of cells. Single-molecule mRNA Fluorescence In Situ Hybridization (FISH) has been established as the standard method for this purpose, but current protocols require a long region of mRNA to be targeted by multiple DNA probes. Here, we introduce a new single-probe FISH protocol termed sFISH for budding yeast, Saccharomyces cerevisiae using a single DNA probe labeled with a single fluorophore. In sFISH, we markedly improved probe specificity and signal-to-background ratio by using methanol fixation and inclined laser illumination. We show that sFISH reports mRNA changes that correspond to protein levels and gene copy number. Using this new FISH protocol, we can detect >50% of the total target mRNA. We also demonstrate the versatility of sFISH using FRET detection and mRNA isoform profiling as examples. Our FISH protocol with single-fluorophore sensitivity significantly reduces cost and time compared to the conventional FISH protocols and opens up new opportunities to investigate small changes in RNA at the single cell level.

PMID:
28666354
PMCID:
PMC5587780
DOI:
10.1093/nar/gkx568
[Indexed for MEDLINE]
Free PMC Article

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