Circulating microRNAs are biomarkers reported to be stable and translational across species. MicroRNA-122 (miR-122) is a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI). We developed a single molecule, dynamic chemical labeling (DCL) assay to directly detect miR-122 in blood. The DCL assay specifically measured miR-122 directly from 10 μL of serum or plasma without any extraction steps, with a limit of detection of 1.32 pM that enabled the identification of DILI. Testing of 192 human serum samples showed that DCL accurately identified patients at risk of DILI after acetaminophen overdose (area under ROC curve 0.98 (95% CI; 0.96–1), P < 0.0001). The DCL assay also identified liver injury in rats and dogs. The use of specific captured beads had the additional benefit of stabilizing miR-122 after sample collection, with no signal loss after 14 days at room temperature, in contrast to PCR that showed significant loss of signal. RNA sequencing demonstrated the presence of multiple miR-122 isomiRs in the serum of patients with DILI that were at low concentration or not present in healthy individuals. Sample degradation over time produced more isomiRs, particularly rapidly with DILI. PCR was inaccurate when analyzing miR-122 isomiRs, whereas the DCL assay demonstrated accurate quantification. We conclude that the DCL assay can accurately measure miR-122 to diagnose liver injury in humans and other species and can overcome microRNA stability and isomiR challenges.

Background Extracellular microRNAs enter kidney cells and modify gene expression. We used a Dicer-hepatocyte-specific microRNA conditional-knock-out (Dicer-CKO) mouse to investigate microRNA transfer from liver to kidney. Methods Dicer flox/flox mice were treated with a Cre recombinase-expressing adenovirus (AAV8) to selectively inhibit hepatocyte microRNA production (Dicer-CKO). Organ microRNA expression was measured in health and following paracetamol toxicity. The functional consequence of hepatic microRNA transfer was determined by measuring the expression and activity of cytochrome P450 2E1 (target of the hepatocellular miR-122), and by measuring the effect of serum extracellular vesicles (ECVs) on proximal tubular cell injury. In humans with liver injury we measured microRNA expression in urinary ECVs. A murine model of myocardial infarction was used as a non-hepatic model of microRNA release. Findings Dicer-CKO mice demonstrated a decrease in kidney miR-122 in the absence of other microRNA changes. During hepatotoxicity, miR-122 increased in kidney tubular cells; this was abolished in Dicer-CKO mice. Depletion of hepatocyte microRNA increased kidney cytochrome P450 2E1 expression and activity. Serum ECVs from mice with hepatotoxicity increased proximal tubular cell miR-122 and prevented cisplatin toxicity. miR-122 increased in urinary ECVs during human hepatotoxicity. Transfer of microRNA was not restricted to liver injury –miR-499 was released following cardiac injury and correlated with an increase in the kidney. Interpretation Physiological transfer of functional microRNA to the kidney is increased by liver injury and this signalling represents a new paradigm for understanding the relationship between liver injury and renal function. Funding Kidney Research UK, Medical Research Scotland, Medical Research Council.

Circulating microRNAs are biomarkers reported to be stable and translational across species. 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 and amplification--free detection method for measuring miR--122 that has translational utility 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 probe that contained a reactive amine instead of a nucleotide at a specific position in the sequence. The single molecule DCL assay specifically measured miR--122 directly from 10 μL of serum or plasma without any extraction steps, with a fit--for--purpose limit of detection of 1.32 pM. In 192 human serum samples, DCL accurately identified patients at risk of DILI (area under ROC curve 0.98 (95%CI 0.96--1), P<0.0001). The miR--122 assay also quantified liver injury in rats and dogs. When DCL beads were added to serum, the miR--122 signal was stabilised (no loss of signal after 14 days at room temperature). By contrast, there was substantial degradation of miR--122 in the absence of beads (≈60% lost in 1 day). RNA sequencing demonstrated the presence of multiple miR--122 isomiRs with DILI that were at low concentration or not present in healthy patient serum. Sample degradation over time produced more isomiRs, particularly rapidly with DILI. PCR was inaccurate when analysing miR--122 isomiRs, whereas the DCL assay demonstrated accurate quantification. In summary, the DCL assay can accurately measure miR--122 directly from serum and plasma to diagnose liver injury in humans and other species, and can overcome important microRNA biomarker analytical and biological challenges.