Single-Probe Fluorescence In Situ Hybridization (Fish) In Budding Yeast

Quantitative determination of the copy number of RNA transcripts in single cells is crucial to understand the genotype-phenotype connection. Current single-molecule Fluorescence In Situ Hybridization (FISH) protocols commonly used for this purpose require a large number of fluorophores per target RNA for single RNA detection, thus limiting the length range of RNA that can be probed. We have developed a FISH protocol for budding yeast, which can detect RNA molecules with a singly labeled 24-nucleotide DNA probe. Our single-probe protocol features highly inclined illumination and methanol fixation. We demonstrated high signal-to-noise and specificity of our protocol when tested against both constitutive and inducible genes in budding yeast. The technique presented offers a cost effective and efficient means of quantifying short RNA transcripts at the single cell level.