Home › Cell Biology › Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis
Cell Biology JoVE (Open Access) Citable · DOI

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

DOI: 10.3791/57774-v
What you'll learn
  • ✓ Perform single-molecule FISH sample preparation in budding yeast cells
  • ✓ Execute hybridization and washing protocols for RNA quantification
  • ✓ Analyze individual mRNA molecules and isoforms in single cells
  • ✓ Quantify RNA abundance during vegetative growth and meiosis
Protocol

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Difficulty
advanced
Total time
~4–6 hours per sample batch (fixation through imaging prep)
Model organism
Saccharomyces cerevisiae (budding yeast)
Biosafety
BSL-1

Steps

1
Fix and digest yeast cell samples

Fix budding yeast cells and treat with digestion enzymes to permeabilize cell walls and enable probe penetration. This step prepares cells for subsequent hybridization.

▶ 00:40
2
Hybridize fluorescent probes to target RNA

Incubate fixed cells with fluorescently labeled oligonucleotide probes targeting specific mRNA sequences. Probes bind to complementary RNA molecules in single cells.

▶ 03:50
3
Wash cells and prepare for microscopy imaging

Remove unbound probe and prepare samples for fluorescence microscopy. Mounting and optimization ensure visualization of individual hybridized RNA molecules.

▶ 05:03
4
Detect and quantify individual mRNA molecules

Image cells by fluorescence microscopy to visualize single mRNA molecules and their isoforms. Analyze signal intensity and molecule count per cell.

▶ 07:03
💬 Comments coming soon