A sensitive one-pot ROA assay for rapid miRNA detection

• DOI: 10.1007/s42994-024-00140-0
• Publication Date: 2024-03-18T12:01:32Z
• Authors: Zhihao Hou; Wenpeng Deng; Alun Li; Ya Zhang; Jianye Chang; Xinyue Guan; Yuxiao Chang; Kaile Wang; Xinjie Wang; Jue Ruan
• Keywords: 0303 health sciences; 03 medical and health sciences; Research Article

AbstractMicroRNAs (miRNAs) and short RNA fragments (18–25 nt) are crucial biomarkers in biological research and disease diagnostics. However, their accurate and rapid detection remains a challenge, largely due to their low abundance, short length, and sequence similarities. In this study, we report on a highly sensitive, one-step RNA O-circle amplification (ROA) assay for rapid and accurate miRNA detection. The ROA assay commences with the hybridization of a circular probe with the test RNA, followed by a linear rolling circle amplification (RCA) using dUTP. This amplification process is facilitated by U-nick reactions, which lead to an exponential amplification for readout. Under optimized conditions, assays can be completed within an hour, producing an amplification yield up to the microgram level, with a detection limit as low as 0.15 fmol (6 pM). Notably, the ROA assay requires only one step, and the results can be easily read visually, making it user-friendly. This ROA assay has proven effective in detecting various miRNAs and phage ssRNA. Overall, the ROA assay offers a user-friendly, rapid, and accurate solution for miRNA detection.