P-GeT Assay: An Innovative Frontier in CircRNA Enrichment

Abstract

Abstract Circular RNAs have garnered attention as essential regulators of gene expression and potential biomarkers in various biological processes and diseases. However, their reliable enrichment from complex RNA pool remains a critical challenge and a costlier approach. Here, we present the "Plug-Gel Trap (P-GeT) assay," as a novel, cost-efficient, and innovative way that bypass the existing exorbitant techniques for circRNA enrichment. By greatly modifying the existing (circular DNA) gel-trap method, the principles of P-GeT assay capitalize on efficient entrapment of unique covalently-closed circular characteristics of RNAs while excluding linear counterparts. In this article, we sketch the fundamental principles behind the P-GeT assay, detailing its workflow and highlighting specificity and efficiency in aggrandizing circRNAs. We compared the competence of two regularly used gel sieves composed of either acrylamides or agarose to significantly trap circular transcripts. The selective trapping by P-GeT assay is validated using the divergent and convergent oligo-nucleotides specific to circular and linear transcripts respectively, and subsequently by northern hybridization. Both the validations are greatly able to detect only the circular form rather than linear transcript. Our study not only introduces an innovative approach for circRNA enrichment but also underscores its versatility and cost-effectiveness, making it accessible to a broader research community. The P-GeT assay represents a significant step towards advancing circRNA studies, unlocking their full potential in deciphering complex biological processes and disease mechanisms.