Imaging and quantification of human and viral circular RNAs

Area of Science:

• Molecular Biology
• Genetics
• Virology

Background:

• Circular RNAs (circRNAs) play crucial roles in gene regulation but are challenging to detect and quantify.
• Existing methods often lack specificity or sensitivity for distinguishing circRNAs from linear RNAs.
• Accurate detection of viral circRNAs is essential for understanding viral pathogenesis.

Purpose of the Study:

• To develop and validate a robust method for cellular detection, imaging, and quantification of circRNAs.
• To differentiate between human and viral circRNAs and their linear counterparts.
• To enable simultaneous visualization and quantification of circRNAs and linear RNAs within single cells.

Main Methods:

• Utilized amplified fluorescence in situ hybridization (ampFISH) combined with single-molecule FISH (smFISH).
• Employed hairpin probes that initiate a target-dependent hybridization chain reaction (HCR) for signal amplification.
• Performed specificity tests including RNase R treatment, probe mismatch analysis, and negative strand probes.

Main Results:

• Successfully detected and imaged human and viral circRNAs (SARS-CoV-2, HCMV) in individual cells.
• Achieved accurate quantification of circRNA molecules using computational image processing.
• Demonstrated high specificity of ampFISH for circRNAs, confirmed by rigorous validation experiments.

Conclusions:

• The developed ampFISH technique provides a sensitive and specific tool for circRNA analysis in cellular and viral contexts.
• This method allows simultaneous detection of circRNAs and their linear counterparts at single-molecule resolution.
• Enables further investigation into circRNA biogenesis, localization, and function.