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Updated: Oct 15, 2025
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Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae
Published on: June 14, 2013
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Visualization and Quantification of Subcellular RNA Localization Using Single-Molecule RNA Fluorescence In Situ
Ankita Arora1, Raeann Goering1,2, Pedro Tirado Velez2
1Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
Methods in Molecular Biology (Clifton, N.J.)
|October 25, 2021
Summary
This study details a single-molecule fluorescent in situ hybridization (smFISH) protocol for visualizing RNA regulation. It presents three probe synthesis methods and automated spot detection for enhanced RNA imaging analysis.
Area of Science:
- Molecular Biology
- Biotechnology
- Cell Biology
Background:
- Advanced imaging techniques enable high-resolution studies of RNA dynamics.
- Understanding RNA regulation requires precise visualization of RNA molecules in situ.
Purpose of the Study:
- To present a comprehensive protocol for single-molecule fluorescent in situ hybridization (smFISH).
- To compare three distinct fluorescent probe synthesis strategies for smFISH.
- To introduce automated spot detection for smFISH data analysis.
Main Methods:
- Protocol development for smFISH.
- Implementation of three probe synthesis methods: commercial, smiFISH, and in-house enzymatic labeling.
- Application of FISH-quant software for automated smFISH spot detection.
Main Results:
- Successful implementation of smFISH with three probe synthesis approaches.
- Demonstration of technical and economic flexibility in probe selection.
- Validation of automated FISH-quant software for accurate spot counting.
Conclusions:
- The described smFISH protocol offers versatile options for RNA molecule imaging.
- Automated detection significantly enhances the efficiency and accuracy of smFISH analysis.
- This methodology provides a robust framework for studying RNA regulation at the single-molecule level.
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