*2.5. Messenger RNA and microRNA Analysis*

miRNAs were extracted from 500 μL human- or 150 μL mouse-plasma by use of the Nucleospin® miRNA Plasma kit (Macherey-Nagel, Düren, Germany), using the manufacturer's protocol. Caenorhabditis elegans miRNA-39 (Cel-miRNA-39) (Qiagen, Hilden, Germany) was added into the plasma lysate before the start of the extraction protocol and served as an external processing control. Total RNA from liver tissue and cultured hepatic stellate cells was extracted by the TRIzol reagent (ThermoFisher scientific, Waltham, USA) and ReliaPrepTM RNA Miniprep system

(Promega, Madison, WI, USA), using the manufacturers' protocol. Messenger RNA (mRNA) was reverse-transcribed into complementary DNA (cDNA) using a mixture of hexamer random primers, Moloney murine leukemia virus reverse transcriptase (M-MLV RT) Buffer, deoxyribonucleotide triphosphate (dNTP) mix, M-MLV RT RNase (H-) Point mutant, and RNasin® Plus RNase Inhibitor (Promega). MicroRNA (miRNA) was reverse transcribed into cDNA using the miScript II RT kit (Qiagen, Hilden, Germany). The expression profiles of selected mRNA and miRNAs were analyzed by quantitative real-time polymerase chain reaction (qPCR), using the GoTaq qPCR Master Mix with BRYT green (Promega) and the miScript SYBR Green PCR kit (Qiagen), respectively, in the QuantStudio 3 real-time PCR system (ThermoFisher scientific). Obtained results were analyzed using the QuantStudio 3 Design and Analysis Software (ThermoFisher scientific). Individual gene and miRNA expression was normalized to Gapdh, RNU6, or Cel-miRNA-39, as appropriate. Relative expression was calculated using the comparative Ct method (2−ΔΔCT). miRNA- (Supplementary Table S1) and gene- (Supplementary Table S2) specific primers were produced by Integrated DNA Technologies (IDT, Leuven, Belgium).
