Femtomolar-level PCR-free quantification of microRNA cancer biomarkers in serum

Abstract

AbstractWe developed a technology to measure microRNA (miRNA) copies in serum and tested it on a commercially available combined human serum (H6914 from Sigma-Aldrich). Copies of miR-15b and miR-16, believed to be constant among healthy and diseased individuals, were measured and agreed with the ones reported by Mitchell PS et al. (2008). Cancer biomarkers let7-b, miR-21, miR-141 and miR-375 varied 3,000 to 6,000 copies per 1 microLiter H6914 (5-10 femtomolar (fM)). Detection and quantification of oligos and miRNAs at such low concentration was shown earlier. It is accomplished by repurposing the commercial MinION nanopore platform to conduct single-molecule voltage-driven ion-channel measurements, employing osmium-tagged oligo probes, and using a publicly available algorithm. These miRNAs were quantified in the serum of healthy individuals or cancer patients using novel optimized probes and a detailed protocol that delivers miRNA copies with better than 85% confidence across all concentrations. A linear correlation, the same with healthy and cancer serum samples, is observed between miR-15b copies and RNA concentration in serum. The assay’s simplicity, readiness, sensitivity, and precision advocate for its use as a Laboratory Developed Test (LDT) for disease-screening based on miRNA dysregulation.