Profiling miRNA Expression in Viral Hepatitis

In order to evaluate miRNA expression levels and profiles of AHE, CHE groups, and HEV-negative control group, analysis of 180 miRNA species was performed using miRCURY LNA Universal cDNA Synthesis Kit II (Exiqon, Vedbaek, Denmark) for reverse transcription, ExiLENT SYBR Green master mix (Exiqon, Vedbaek, Denmark) for quantitative PCR (qPCR) amplification and Serum/Plasma Focus qPCR Panels V4.RO (Exiqon, Vedbaek, Denmark) according to the manufacturer´s instructions. As internal amplification controls synthetic miRNAs UniSp2, 4 and 5 were added as spike-ins before extraction and UniSp6 before cDNA synthesis (Exiqon, Vedbaek, Denmark). Data analysis was performed with GenEx qPCR analysis software (Exiqon, Vedbaek, Denmark). Raw cycle threshold (Ct) values were inter-plate calibrated using UniSp3. To identify endogenous reference miRNAs and regulated miRNAs for expression analyses during HEV infection, global mean normalization was performed. In brief, fold changes (FC) of miRNA expression in AHE and CHE patients compared to controls were calculated using the 2^−ΔΔCt method. Four regulated miRNAs were selected for further investigation based on previous studies^{24 (link)–30 (link)}. The three most stably expressed miRNAs were selected as endogenous reference genes.