smiFISH and FISH-quant - a flexible single RNA detection approach with super-resolution capability

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Single molecule FISH (smFISH) is crucial for studying gene expression and RNA localization at the individual mRNA level within cells.
• Existing smFISH methods can be costly and require custom probe synthesis for each experiment.

Purpose of the Study:

• To develop a cost-effective, flexible, and highly efficient RNA visualization and quantification method.
• To enable multicolor smFISH and super-resolution imaging without extensive probe re-design.
• To provide automated software for probe design and image analysis.

Main Methods:

• Developed single molecule inexpensive FISH (smiFISH) using unlabelled primary probes and labelled secondary detector oligonucleotides.
• Demonstrated multicolor labeling by switching secondary detectors with the same primary probes.
• Integrated smiFISH with expansion microscopy using acrydite detector oligonucleotides for 3D super-resolution imaging.
• Created automated software for probe design and quantitative analysis of smFISH images.

Main Results:

• smiFISH significantly increases mRNA detection efficiency due to the use of more probes per target.
• The method allows flexible multicolor RNA detection without synthesizing new probe sets.
• Combined smiFISH with expansion microscopy to resolve RNA transcripts in 3D below the diffraction limit.
• Automated software streamlines the entire workflow from probe design to data analysis.

Conclusions:

• smiFISH provides a powerful, accessible, and versatile workflow for single-cell RNA analysis.
• The technique facilitates high-efficiency, multicolor, and super-resolution imaging of RNA.
• Automated tools simplify and enhance the quantitative analysis of RNA molecules in single cells.