Single-Molecule RNA Fluorescence In Situ Hybridization (smRNA-FISH) Labeling and Imaging of mRNA to Determine Subcellular Localization.

The protocol reported in this chapter describes methodologies for labeling of RNA by single-molecule RNA fluorescence in situ hybridization (smRNA-FISH), performing imaging confocal microscopy to image RNAs, and post-acquisition analyses. Specifically, we describe methods to design and generate smRNA-FISH probes, label RNAs in situ, and image RNAs. We also describe stepwise post-acquisition analyses using Image J to determine the absolute quantity, subcellular localization, and translation status of messenger RNAs. We provide examples of labeling full-length RNAs and their subspecies, quantifying RNA localization to organelles and ribonucleoprotein granules, and analyses of the translation status of individual mRNAs.