HT-smFISH: a cost-effective and flexible workflow for high-throughput single-molecule RNA imaging.

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• Single-molecule fluorescence in situ hybridization (smFISH) is crucial for visualizing RNA in its native cellular environment, advancing gene expression and cellular function studies.
• A significant limitation of smFISH is the high cost and difficulty in performing high-throughput experiments due to expensive probe sets.

Purpose of the Study:

• To develop a simple, cost-efficient, and scalable method for high-throughput smFISH (HT-smFISH).
• To enable the imaging of hundreds to thousands of single endogenous RNA molecules in a 96-well plate format.

Main Methods:

• HT-smFISH utilizes RNA probes generated in vitro from unlabeled oligonucleotide pools, replacing costly commercial DNA probe sets.
• The protocol integrates oligo pool design, probe set generation, optimized hybridization, and image acquisition/analysis guidelines.
• The method is achievable within approximately 20 days using standard molecular biology techniques and fluorescence microscopy.

Main Results:

• HT-smFISH significantly reduces the cost per targeted RNA compared to traditional smFISH methods.
• The method is scalable and flexible, allowing for the generation of individual probes for numerous RNA species.
• Successfully demonstrated imaging of thousands of single endogenous RNA molecules in a 96-well plate format.

Conclusions:

• HT-smFISH provides a practical solution for medium- to high-throughput RNA imaging at single-molecule sensitivity.
• This method facilitates systematic screens and broadens the application of smFISH in biological research.
• The developed protocol lowers the barrier for high-throughput RNA analysis, making it more accessible.