Diagnostic and prognostic role of circular RNAs: Analytical challenges and emerging opportunities

Circular RNAs (circRNAs) are covalently closed non-coding RNAs characterized by high stability, tissue-specific expression, and important regulatory roles in gene expression and cellular pathways across cancer, neurodegenerative, cardiovascular, autoimmune, and infectious diseases. Their stability in biofluids and disease-associated expression patterns make them attractive candidates for diagnostic and prognostic biomarkers. This review examines the analytical strategies used to detect and quantify circRNAs, including amplification-based methods (RT-qPCR, droplet digital PCR, and isothermal amplification), hybridization-based assays (Northern blot, FISH, and microarrays), sequencing approaches (short- and long-read RNA-seq), and emerging biosensor technologies. These methods are discussed in the context of their principles, strengths, limitations, and suitability for different research and clinical applications. By integrating current knowledge of circRNA biology with practical guidance on analytical workflows, this review provides a framework for selecting appropriate detection strategies and supports the development of reliable circRNA-based diagnostics and future precision medicine applications.