MicroRNAs(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 abundanc...MicroRNAs(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.展开更多
基金supported the National Key R&D Program of China(2019YFA0707003 and 2022YFC3400300 to J.R.)the Innovation Program of Chinese Academy of Agricultural Sciences.
文摘MicroRNAs(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.
