Related Experiment Video
Updated: May 8, 2026
08:18
Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes
Published on: April 24, 2019
Single-molecule mRNA detection and counting in mammalian tissue
Anna Lyubimova1, Shalev Itzkovitz, Jan Philipp Junker
1Department of Physics, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Nature Protocols
|August 17, 2013
Summary
This study introduces a new method to visualize and quantify single messenger RNA (mRNA) molecules in mammalian tissues using fluorescently labeled oligonucleotide probes and microscopy. The protocol allows for precise localization of mRNA within individual cells for detailed analysis.
Area of Science:
- Molecular Biology
- Biotechnology
- Cell Biology
Background:
- Accurate visualization and quantification of single mRNA molecules are crucial for understanding gene expression dynamics in tissues.
- Existing methods may lack the resolution or specificity required for single-molecule detection within complex tissue architectures.
- Developing robust protocols for in situ mRNA analysis is essential for advancing fields like developmental biology and disease research.
Purpose of the Study:
- To establish a detailed protocol for the visualization and quantification of single mRNA molecules in mammalian tissues.
- To enable precise spatial mapping of mRNA expression at the single-cell level within tissue sections.
- To provide a freely available computational tool for automated spot detection and cell assignment of mRNA molecules.
Main Methods:
- Hybridization of sets of ~50 fluorophore-labeled oligonucleotide probes to target mRNAs in fixed tissue sections.
- Detection of hybridized probes as diffraction-limited spots using fluorescence microscopy.
- Counterstaining tissue sections with DNA dyes (e.g., DAPI) and antibody-coupled dyes for visualizing nuclei and cell borders.
- Automated spot detection and semi-automated assignment of mRNA spots to individual cells using custom software and a graphical user interface.
Main Results:
- Successful visualization and quantification of single mRNA molecules in mouse and human tissues.
- Demonstration of precise localization of mRNA within specific cell types and tissues, exemplified in mouse small intestine.
- Development and availability of custom software for automated data processing, facilitating reproducible analysis.
- The entire procedure, from tissue dissection to data acquisition, can be completed in 3 days, with data analysis taking an additional 3-7 days.
Conclusions:
- The presented protocol offers a powerful and accessible method for high-resolution in situ analysis of mRNA expression in mammalian tissues.
- This technique facilitates quantitative studies of gene expression at the single-cell and subcellular levels, advancing our understanding of cellular function and heterogeneity.
- The freely available software and user-friendly interface lower the barrier for researchers to perform advanced mRNA localization and quantification experiments.
