RNA modifications are essential regulators of gene expression and cellular function, modulating RNA stability, splicing, translation, and localization. Dysregulation of these modifications has been linked to cancer, neurodegenerative disorders, viral infections, and other diseases. Precise quantification and mapping of RNA modifications are crucial for understanding their biological roles. This review summarizes current and emerging methodologies for RNA modification analysis, including mass spectrometry, antibody-based and non-antibody-based approaches, PCR- and NMR-based detection, chemical- and enzyme-assisted sequencing, and nanopore direct RNA sequencing. We also highlight advanced techniques for single-cell and single-molecule imaging, enabling the study of modification dynamics and cellular heterogeneity. The advantages, limitations, and challenges of each method are discussed, providing a framework for selecting appropriate analytical strategies. Future perspectives emphasize high-throughput, multiplexed, and single-cell approaches, integrating multiple technologies to decode the epitranscriptome. These approaches form a robust toolkit for uncovering RNA modification functions, discovering biomarkers, and developing novel therapeutic strategies.
Tian et al. (2025) studied this question.