Duplex-specific nuclease-assisted isothermal signal amplification coupled with HPLC-UV for simultaneous detection of multiple microRNAs

Abstract

MicroRNAs (miRNAs) regulate post-transcriptional gene expression by specifically recognizing mRNAs and have emerged as potential biomarkers for many diseases. To improve the sensitivity of disease diagnosis, simultaneous detection of multiple miRNAs is highly required. However, the conventional stem–loop reverse transcription qPCR (RT-qPCR) method cannot achieve multiplexed miRNA detection in a single run. To address these limitations, we developed a multiplexed miRNA detection method based on duplex-specific nuclease (DSN)-assisted isothermal signal amplification with HPLC coupled with UV. In this strategy, target miRNAs trigger DSN enzyme activity and hydrolysis of polyadenine (poly A) and polyguanine (poly G) sequences for the release of A and G, and this is followed by chromatographic separation and quantitative detection. The proposed method enabled the detection of target miRNAs ranging from 500 fM to 500 nM and could discriminate homologous family members and sequences with 2- or 4-base mismatches. Finally, the method was successfully applied to detect miRNAs in total RNA from MCF-7 cells, and the results were consistent with those obtained by conventional stem–loop RT-qPCR, demonstrating the favorable accuracy of the newly developed method. Based on this method, the introduction of additional DNA probes containing polydeoxythymidine (poly T) and polydeoxycytidine (poly C) sequences enables the simultaneous analysis of 4 miRNAs, thereby demonstrating considerable application potential.