Simultaneous dual-microRNA detection <i>via</i> magnetic separation-assisted Duplex DNase signal amplification in a single reaction

MicroRNAs (miRNAs) are promising diagnostic biomarkers, but non-canonical miRNA-mRNA pairing causes co-dysregulation in disease, reducing single-miRNA specificity. Thus, the detection of multiple miRNAs helps reduce the false positive rate in disease diagnosis. However, stem-loop RT-qPCR, the current gold standard for miRNA detection, can only assay one miRNA per reaction. When multiple miRNAs need to be detected, this method exhibits a critical limitation: low throughput. We developed a one-reaction fluorescence method enabling simultaneous dual-miRNA quantification. This method utilizes DNA probes that can specifically recognize target miRNAs. These probes are dual-labeled with biotin and fluorescent molecules (FAM and Cy3). Leveraging the specific DNA strand cleavage activity of Duplex DNase and the ability of streptavidin-conjugated magnetic nanoparticles to capture biotinylated probes, we detect the fluorescently labeled oligonucleotides remaining in the reaction solution using different excitation and emission wavelengths, thereby indirectly detecting two types of miRNAs. The dual-miRNA detection method developed in this study is capable of detecting miR-21 and miR-141 at a minimum concentration of 10 fM. This approach enhances detection efficiency and facilitates comprehensive multi-miRNA profiling.