Direct detection of circulating microRNA-122 using dynamic chemical labelling with single molecule detection overcomes stability and isomiR challenges for biomarker qualification

ABSTRACT: Circulating microRNAs are biomarkers reported to be stable and translational acrossspecies. miR-122 (miR-122-5p) is a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI). Our objective was to develop an extraction-free andamplification-free detection method for measuring miR-122 that has translationalutility in context of DILI. We developed a single molecule dynamic chemical labelling(DCL) assay based on miR-122 hybridization to an abasic peptide nucleic acid probethat contained a reactive amine instead of a nucleotide at a specific position in thesequence. The single molecule DCL assay specifically measured miR-122 directly from10 μL of serum or plasma without any extraction steps, with a fit-for-purpose limit ofdetection of 1.32 pM. In 192 human serum samples, DCL accurately identified patientsat risk of DILI (area under ROC curve 0.98 (95%CI 0.96-1), P<0.0001). The miR-122assay also quantified liver injury in rats and dogs. When DCL beads were added toserum, the miR-122 signal was stabilised (no loss of signal after 14 days at roomtemperature). By contrast, there was substantial degradation of miR-122 in the absenceof beads (≈60% lost in 1 day). RNA sequencing demonstrated the presence of multiplemiR-122 isomiRs with DILI that were at low concentration or not present in healthypatient serum. Sample degradation over time produced more isomiRs, particularlyrapidly with DILI. PCR was inaccurate when analysing miR-122 isomiRs, whereas theDCL assay demonstrated accurate quantification. In summary, the DCL assay canaccurately measure miR-122 directly from serum and plasma to diagnose liver injury inhumans and other species, and can overcome important microRNA biomarker analyticaland biological challenges.

ORGANISM(S): Homo sapiens

PROVIDER: GSE140833 | GEO | 2019/11/23

REPOSITORIES: GEO