Optimized protocol for single-molecule RNA FISH to visualize gene expression in S. cerevisiae.

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Single-molecule RNA fluorescence in situ hybridization (smFISH) enables subcellular RNA analysis.
• smFISH provides spatial and intensity data to differentiate RNA states, reflecting transcriptional activity.
• Existing smFISH protocols require optimization for specific model organisms like yeast.

Purpose of the Study:

• To present an optimized protocol for smFISH in Saccharomyces cerevisiae.
• To improve the homogeneity and reliability of smFISH results in yeast.
• To facilitate detailed analysis of RNA dynamics in yeast cells.

Main Methods:

• Optimization of lyticase digestion time for Saccharomyces cerevisiae cell permeabilization.
• Refinement of hybridization steps for enhanced probe binding and signal detection.
• Application of smFISH for visualizing endogenous RNA molecules at the single-molecule level.

Main Results:

• Achieved more homogenous smFISH results in Saccharomyces cerevisiae.
• Demonstrated successful subcellular visualization and localization of endogenous RNA.
• Enabled quantification of individual RNA molecules, distinguishing nascent and mature transcripts.

Conclusions:

• The optimized smFISH protocol provides a robust method for yeast RNA analysis.
• This technique allows for precise measurement of transcriptional activity in yeast.
• The protocol facilitates deeper understanding of gene expression dynamics in a model eukaryote.