A Tailored Workflow for Circular RNA Enrichment and Analysis from Human Whole Blood

Abstract

CircRNAs are covalently closed ncRNAs originating through back splicing; their expression is finely regulated, displaying specific patterns across different cell types, tissues, and developmental stages. While the molecular functions of circRNAs are not completely elucidated, their regulatory involvement in physiological processes is well established, alongside their dysregulation in several human disorders. These features, together with their higher stability compared to other ncRNAs, make this class of molecules promising theragnostic agents, particularly in biomarker discovery. Accordingly, it is crucial to develop and standardize experimental strategies that improve circRNA analysis, ensuring accurate and effective isolation of these molecules. In biomarker discovery, selecting the appropriate biological matrix is critical; whole blood is often preferred for its accessibility and minimally invasive collection. Because circRNAs are present in human peripheral blood and show promise as disease theragnostic biomarkers, we established a preliminary workflow tailored to isolate and analyze circRNAs from whole blood. The promising effectiveness and robustness of this workflow were demonstrated by qPCR analysis, suggesting highly reproducible detection and reliability in isolating and analyzing circRNAs. This, together with their stability and specific expression profiles, supports the utility of circRNAs in biomarker discovery and advanced circRNA research and contributes to accelerating their future integration into theragnostic applications in clinical settings.