Single-Molecule Resolution Fluorescent In Situ Hybridization (smFISH) in the Yeast S. cerevisiae
Regulating gene expression is a major task for all cellular systems. RNA production and degradation plays a critical role in this process and accurately measuring cellular mRNA levels is essential to understanding gene expression regulation. Classical biochemical assays that study gene expression rely on extracting RNAs from large populations of cells, taking them out of their native context and thereby losing spatial information as well as cell-to-cell variability. In this chapter, we describe a fluorescent in situ hybridization (FISH) technique that circumvents this problem by detecting single RNAs in single cells. The technique employs multiple single-stranded short DNA probes fluorescently labeled with organic dyes that hybridize to target RNAs in fixed cells, allowing quantification and localization of RNAs at the single-cell level and at single-molecule resolution. The protocol described here has been optimized for the yeast S. cerevisiae.
Key wordsGene expression Single-cell imaging Single RNA resolution fluorescent in situ hybridization Yeast mRNA detection
We thank Marlene Oeffinger as well as members of the Zenklusen laboratory for comments and discussions on the manuscript. The laboratory of Daniel Zenklusen is supported by the Canadian Institutes of Health Research (MOP-BMB-232642), the Natural Sciences and Engineering Research Council of Canada, the Fonds de recherche Santé Québec, and the Canada Foundation for Innovation.