Single-molecule Fluorescence in situ Hybridization (smFISH) for RNA Detection in Adherent Animal Cells

Gal Haimovich; Jeffrey E Gerst

doi:10.21769/BioProtoc.3070

Single-molecule Fluorescence in situ Hybridization (smFISH) for RNA Detection in Adherent Animal Cells

Bio Protoc. 2018 Nov 5;8(21):e3070. doi: 10.21769/BioProtoc.3070.

Authors

Gal Haimovich¹, Jeffrey E Gerst¹

Affiliation

¹ Dept. of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.

Abstract

Transcription and RNA decay play critical roles in the process of gene expression and the ability to accurately measure cellular mRNA levels is essential for understanding this regulation. Here, we describe a single-molecule fluorescent in situ hybridization (smFISH) method (as performed in Haimovich et al., 2017 ) that detects single RNA molecules in individual cells. This technique employs multiple single-stranded, fluorescent labeled, short DNA probes that hybridize to target RNAs in fixed cells, allowing for both the quantification and localization of cytoplasmic and nuclear RNAs at the single-cell level and single-molecule resolution. Analyzing smFISH data provides absolute quantitative data of the number of cytoplasmic ("mature") mRNAs, the number of nascent RNA molecules at distinct transcription sites, and the spatial localization of these RNAs in the cytoplasm and/or nucleoplasm.

Keywords: Fluorescence in situ hybridization; Adherent cells; Fluorescence microscopy; Single molecule resolution; Transcription; mRNA.