Simple Isothermal Strategy for Multiplexed, Rapid, Sensitive, and Accurate miRNA Detection

Abstract

MicroRNAs (miRNA) are potential biomarkers. Current preferred miRNA detection methods rely on various PCR-based approaches. Although effective, such methods are typically tedious, slow, and require expensive equipment. Hence, faster and simpler miRNA detection approaches are still needed. Herein, we describe miRPA: a novel combination of recombinase polymerase amplification (RPA) with PBCV-1 DNA ligase for simple, specific, rapid, and multiplexed isothermal miRNA detection. MiRPA is sensitive to picogram levels of total RNA input (or ∼40 copies/pg) and can discriminate between closely related miRNAs. MiRPA was applied to cell lines and was subsequently validated with a commercial qPCR method. Potential clinical application was also demonstrated by detecting miRNAs in urine-derived RNA. This is the first application of RPA for rapid isothermal miRNA detection, and it could have wide applications as a miRNA sensor in both research and in the clinic.