METHOD FOR DIAGNOSING HEPATIC PATHOLOGIES IN DOGS USING CFA-MIRNAS Russian patent published in 2021 - IPC C12Q1/68 C12N15/00 

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of veterinary science and biotechnology. Proposed is a method for using circulating caninefamiliaris (cfa) microRNAs (miRNAs) produced from hepatocytes as new non-invasive diagnostic biomarkers for accurately reflecting the type of ongoing hepatic pathologies in dogs with different morphologies of extrahepatic and intrahepatic congenital portosystemic shunts (ICPS). The method is implemented by extracting the total RNA from serum samples of dogs with the confirmed presence of ICPS and various hepatic pathologies, followed by reverse transcription with subsequent amplification in RT-PCR in real time using canine-specific primers for cfa-miR-122-5p, cfa-miR-34a-5p, cfa-miR-21-5p and cfa-miR-126-5p. All analyses were applied in three repeats for each sample, and the resulting average cycle threshold (Ct) for each tested microRNA was normalised relative to the average Ct of cfa-miRNA-16 (stably expressed endogenous control) and cel-miRNA-39 (exogenous spike in the control). The relative multiple change in each tested cfa-miRNA in the groups of ICPS compared to the corresponding healthy controls was then determined using the 2^-ΔΔ^Ct method. Tests have proved that cfa-miR-122 at a threshold value of ≥1.32 to 1.45 constitutes a diagnostic tool for microvesicular steatosis. Cfa-miR-34a at a threshold value of ≥ 1.15 to 1.23 constitutes a diagnostic tool for vacuolated hepatocytes with macrovesicular steatosis, as well as lipid or pigmented granuloma. Cfa-miR-21 at a threshold value of ≥ 1.07 to 1.23 constitutes a diagnostic tool for portal and parenchymal fibrosis. Cfa-miR-126 at a threshold value of ≥ 1.14 to 1.16 constitues a diagnostic tool for changing the hepatic blood supply in the form of arteriole proliferation and duct proliferation associated therewith in dogs with ICPS.

EFFECT: invention provides a possibility of using the selected serum cfa-miRNAs as new non-invasive biomarkers for reflecting the histopathological and molecular events occurring in the liver in each type of shunt with high degree of sensitivity and specificity.

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