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A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae
Published on: July 18, 2011
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mRNA detection in budding yeast with single fluorophores
Gable M Wadsworth1, Rasesh Y Parikh1, John S Choy2
1School of Physics, Georgia Institute of Technology, 837 State Street, Atlanta, GA 30332-0430, USA.
Nucleic Acids Research
|July 2, 2017
Summary
We developed a new single-probe Fluorescence In Situ Hybridization (sFISH) method for quantifying mRNA in single cells. This cost-effective technique improves accuracy and reduces time compared to existing methods.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Quantitative measurement of single-cell mRNA levels is crucial for understanding cellular heterogeneity.
- Conventional single-molecule mRNA Fluorescence In Situ Hybridization (FISH) requires multiple probes targeting long mRNA regions, increasing complexity and cost.
Purpose of the Study:
- To introduce a novel, simplified single-probe FISH (sFISH) protocol for budding yeast (Saccharomyces cerevisiae).
- To enhance probe specificity and signal-to-background ratio for accurate mRNA quantification in single cells.
Main Methods:
- Developed sFISH protocol utilizing a single DNA probe with a single fluorophore.
- Optimized probe specificity and signal-to-background ratio through methanol fixation and inclined laser illumination.
- Validated sFISH by correlating mRNA levels with protein levels and gene copy number.
Main Results:
- sFISH successfully detects over 50% of total target mRNA molecules.
- Demonstrated versatility of sFISH for FRET detection and mRNA isoform profiling.
- Achieved significant reduction in cost and time compared to conventional FISH protocols.
Conclusions:
- sFISH offers a simplified, cost-effective, and time-efficient alternative for single-molecule mRNA quantification.
- This method enhances the ability to investigate subtle RNA changes at the single-cell level.
- sFISH opens new avenues for studying phenotypic variability and gene expression dynamics.
