Multiplex digital PCR for the simultaneous quantification of a miRNA panel

microRNAs (miRNAs) are small non-coding RNAs regulating gene expression. They have attracted significant interest as biomarkers for early diagnosis, prediction and monitoring of treatment response in many diseases. As individual miRNAs often lack the required sensitivity and specificity, miRNA signatures are developed for clinical applications. Digital PCR (dPCR) is a sensitive fluorescent-based quantification method, that can be used to detect the expression of miRNAs in patient samples. Our study presents the first proof-of-concept of a multiplexed dPCR assay for the simultaneous analysis and quantification of multiple miRNAs. After reverse transcription (RT) using a pool of miRNA-specific stem-loop primers, dPCR was performed with a universal reverse primer and miRNA-specific forward primers along with fluorescently-labelled hydrolysis probes. Multiple experimental parameters were evaluated and strategies for modulating the observed signals were devised. The optimised assay was applied to the analysis of miRNAs from cell lines and biological samples. Although absolute quantification was lost, due to the reverse transcription step, quantification was linear for the dilution series and results were highly reproducible for independent dPCR and RT reactions. Our results confirmed the high sensitivity of dPCR for patient samples. We demonstrate the feasibility and reliability of multiplexed detection and quantification of miRNAs by dPCR that can be applied in a clinical setting to evaluate miRNA signatures. • MiRNA signatures represent promising biomarkers for clinical applications. • First proof-of-concept of a multiplexed digital PCR assay for miRNA analysis. • Combination of miRNA-specific stem-loop primers and dPCR with hydrolysis probes. • Linear and reproducible quantification results for up to six miRNAs. • Optimised protocol can be applied to different types of biological samples.