Target-Specific Profiling of RNA m⁵C Methylation Level Using Amplicon Sequencing

Area of Science:

• Molecular Biology
• Epigenetics
• RNA Biology

Background:

• 5-methylcytosine (m⁵C) is a crucial epitranscriptomic modification impacting RNA function.
• Accurate mapping and quantification of m⁵C in cellular RNA are essential for understanding its biological roles.
• Existing methods require refinement for robust m⁵C detection in RNA.

Purpose of the Study:

• To detail a reliable method for detecting and quantifying m⁵C modifications in RNA.
• To adapt the established bisulfite conversion technique for RNA analysis.
• To provide a protocol for locus-specific m⁵C detection.

Main Methods:

• RNA isolation and bisulfite conversion.
• Locus-specific PCR amplification of target RNA regions.
• Next-generation sequencing (Illumina MiSeq) for m⁵C site detection.

Main Results:

• Successful application of bisulfite treatment to RNA samples.
• Demonstration of locus-specific PCR for amplifying modified RNA.
• Validation of sequencing-based detection of m⁵C sites on the Illumina MiSeq platform.

Conclusions:

• The described method offers a robust approach for m⁵C mapping in RNA.
• This technique facilitates precise quantification of RNA m⁵C levels.
• The protocol advances the field of epitranscriptomics by enabling detailed analysis of RNA modifications.