Highly Sensitive Detection of Multiple MicroRNAs by High-Performance Liquid Chromatography Coupled with Long and Short Probe-Based Recycling Amplification.

ABSTRACT

This report demonstrated the utility of high-performance liquid chromatography (HPLC)-fluorescence detection for selective separation and sensitive quantification of multiple microRNAs (miRNAs). A duplex specific nuclease (DSN)-assisted target recycling amplification strategy was developed to enhance the signals of miRNAs, which alleviates the low sensitivity of conventional HPLC to nucleic acids. To separate the signals of different miRNAs, DNA probes with different lengths and base sequences were immobilized on magnetic beads. The application of an effective magnetic separation minimized the background signal and extended the dynamic range. This assay achieved a limit of detection of 0.39 fM for miRNA-122, 0.30 fM for miRNA-155, and 0.26 fM for miRNA-21, respectively. The proposed assay was successfully applied to detect simultaneously miRNA-122, miRNA-155, and miRNA-21 in serum samples from healthy persons and cervical cancer patients, and the results were then compared with those of quantitative real-time-polymerase chain reaction amplification.