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mRNA detection in budding yeast with single fluorophores

Gable M. Wadsworth, Rasesh Y. Parikh, John S. Choy, Harold D. Kim
doi: https://doi.org/10.1101/155143
Gable M. Wadsworth
1School of Physics, Georgia Institute of Technology, 837 State Street, Atlanta, GA 30332-0430
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Rasesh Y. Parikh
1School of Physics, Georgia Institute of Technology, 837 State Street, Atlanta, GA 30332-0430
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John S. Choy
2Department of Biology, The Catholic University of America, 620 Michigan Avenue NE. Washington, DC 20064, U.S.A.
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Harold D. Kim
1School of Physics, Georgia Institute of Technology, 837 State Street, Atlanta, GA 30332-0430
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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 more than 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.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted June 24, 2017.
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mRNA detection in budding yeast with single fluorophores
Gable M. Wadsworth, Rasesh Y. Parikh, John S. Choy, Harold D. Kim
bioRxiv 155143; doi: https://doi.org/10.1101/155143
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mRNA detection in budding yeast with single fluorophores
Gable M. Wadsworth, Rasesh Y. Parikh, John S. Choy, Harold D. Kim
bioRxiv 155143; doi: https://doi.org/10.1101/155143

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