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

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Traditional RNA quantification methods like RT-PCR, microarrays, and sequencing analyze bulk cell populations.
• Population-based methods obscure crucial information regarding spatial RNA distribution and stochastic gene expression within individual cells.
• Understanding single-cell RNA dynamics is vital for deciphering cellular heterogeneity and phenotypic variation.

Purpose of the Study:

• To describe a detailed protocol for Fluorescent in situ hybridization (FISH) enabling single mRNA quantification in budding yeast.
• To highlight FISH's capability in preserving and analyzing the spatial organization and dynamic fluctuations of RNA within individual cells.
• To present FISH as a superior alternative to population-based methods for capturing single-molecule RNA data.

Main Methods:

• Utilizing fluorescently labeled single-stranded DNA probes for hybridization with fixed yeast cells.
• Employing wide-field epifluorescence microscopy for 3D image acquisition of hybridized cells.
• Applying a spot-detection algorithm to identify and quantify diffraction-limited fluorescent spots representing single mRNAs.

Main Results:

• Successful quantification of single mRNA molecules within individual yeast cells.
• Demonstration that FISH preserves the precise spatial localization of mRNAs within the cellular context.
• Observation of stochastic fluctuations in mRNA levels at the single-cell level, which are typically averaged out in population studies.

Conclusions:

• FISH provides a powerful tool for single-molecule mRNA quantification in budding yeast.
• This technique retains critical information on RNA spatial distribution and cellular stochasticity, essential for understanding phenotypic variation.
• FISH offers a significant advantage over bulk RNA analysis methods for detailed cellular insights.