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Updated: Sep 3, 2026

RNA Fluorescence In Situ Hybridization for Long Non-Coding RNA Localization in Human Osteosarcoma Cells
Published on: June 16, 2023
Single-Molecule RNA Fluorescence In Situ Hybridization (smRNA-FISH) Labeling and Imaging of mRNA to Determine
Skyler Briggs1,2,3, James M Burke4,5
1Department of Molecular Medicine, The Herbert Wertheim University of Florida, Scripps Institute for Biomedical Innovation and Technology, Jupiter, FL, USA.
Abstract:
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.
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