A digital PCR-based protocol to detect and quantify RNA editing events at hotspots

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• RNA editing enzymes like APOBEC3A and CRISPR base editors modify RNA transcripts.
• Accurate quantification of RNA editing is essential for understanding enzyme activity and efficiency.
• Existing methods may lack the sensitivity or speed required for precise measurement.

Purpose of the Study:

• To develop a rapid and accurate method for quantifying RNA editing events.
• To enable absolute quantification of RNA editing activity in cellular and patient samples.
• To provide a sensitive tool for assessing RNA editing enzyme performance.

Main Methods:

• Utilized digital PCR (dPCR) for sensitive detection of rare mutations.
• Developed a dPCR assay for absolute quantification of RNA editing events.
• Applied micro-partitioning of bulk PCR reactions for enhanced precision.

Main Results:

• The developed dPCR assay allows for rapid quantification of RNA editing activity.
• Achieved precise absolute quantification of RNA editing events.
• Demonstrated the applicability of the method in cell lines and patient samples.

Conclusions:

• Digital PCR offers a sensitive and quantitative approach to measure RNA editing.
• This method provides a quick and reliable way to assess RNA editing enzyme efficiency.
• The protocol is suitable for diverse biological samples, aiding research in RNA modification.