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

Heta P Patel; Ineke Brouwer; Tineke L Lenstra

doi:10.1016/j.xpro.2021.100647

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

STAR Protoc. 2021 Jul 6;2(3):100647. doi: 10.1016/j.xpro.2021.100647. eCollection 2021 Sep 17.

Authors

Heta P Patel¹, Ineke Brouwer¹, Tineke L Lenstra¹

Affiliation

¹ Division of Gene Regulation, The Netherlands Cancer Institute, Oncode Institute, 1066CX Amsterdam, the Netherlands.

Abstract

Single-molecule RNA fluorescence in situ hybridization (smFISH) allows subcellular visualization, localization, and quantification of endogenous RNA molecules in fixed cells. The spatial and intensity information of each RNA can be used to distinguish mature from nascent transcripts inside each cell, revealing both past and instantaneous transcriptional activity. Here, we describe an optimized protocol for smFISH in Saccharomyces cerevisiae with optimized lyticase digestion time and hybrization steps for more homogenous results. For complete details on the use and execution of this protocol, please refer to Donovan et al. (2019).

Keywords: cell biology; gene expression; in situ hybridization; microscopy; model organisms; molecular biology; single cell; single-molecule assays.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Gene Expression Regulation, Fungal
In Situ Hybridization, Fluorescence / methods*
Molecular Imaging / methods*
RNA Probes / genetics
RNA, Fungal
Saccharomyces cerevisiae / genetics*
Single Molecule Imaging / methods*

Substances

RNA Probes
RNA, Fungal