Highly-sensitive amplification-free analysis of multiple miRNAs by capillary electrophoresis

Sets of deregulated microRNAs (miRNAs), termed miRNA signatures, are promising biomarkers for cancer. Validation of miRNA signatures requires a technique that is accurate, sensitive, capable of detecting multiple miRNAs, fast, robust, and not cost-prohibitive. Direct quantitative analysis of multiple miRNAs (DQAMmiR) is a capillary electrophoresis (CE)-based hybridization assay that was suggested as a methodological platform for validation and clinical use of miRNA signatures. While satisfying the other requirements, DQAMmiR is not sufficiently sensitive to detect low-abundance miRNAs. Here, we solve this problem by combining DQAMmiR with the preconcentration technique, isotachophoresis (ITP). The sensitivity improved 100 times (to 1 pM) allowing us to detect low-abundance miRNAs in an RNA extract. Importantly, ITP-DQAMmiR can be performed in a fully automated mode using a commercial CE instrument making it suitable for practical applications.