Quantification of miR-122:
Serum samples were stored at -80°C before analysis. Freeze-thaw cycles were avoided to preserve miRNA integrity. MiR-122 was quantified in samples from healthy volunteers and the ALISTER study. All ALISTER samples were stored for a maximum of one year (median [IQR]: 16.3 [7.6-33.3] weeks). This is less time in storage than previously published studies which have demonstrated miRNA stability (Balzanoet al. , 2015; Shaughnessy et al. , 2015). MiRNA was extracted using the miRNeasy Serum/Plasma kit (Qiagen, Venlo, Netherlands) following the manufacturer’s instructions. Total RNA was extracted from 50 μL diluted in 150 μL nuclease free water. Briefly, RNA was extracted from the serum by lysis reagent (1000 μL) and chloroform (200 μL). After centrifugation at 12000xg for 15 min at 4°C up to 600 μL of the aqueous phase was transferred to a new tube with 900 μL absolute ethanol. RNA was purified on a RNeasy minElute spin column and eluted in 15 μL RNase-free water and stored at −80°C. Extraction efficiency was monitored by adding 5.6 × 10⁸ copies of synthetic C. elegans miR-39 spike-in control after the addition of lysis reagent before the addition of chloroform and phase separation. The miScript II Reverse Transcription kit was used to prepare cDNA according to the manufacturer’s instructions. Briefly, 2.5 μL of RNA eluate was reverse transcribed into cDNA. The synthesised cDNA was diluted and used for cDNA template in combination with the miScript SYBR Green PCR kit (Qiagen, Venlo, The Netherlands) using the specific miScript assays (Qiagen, Venlo, The Netherlands). RT-PCR was performed in duplicate on a Light Cycler 480 (Roche, Burgess Hill, UK) using the recommended miScript cycling parameters. In this study, miR-122 was quantified in fM by generating a standard curve. Serial dilutions of known standard were made using synthetic miR-122 (syn hsa-miR-122-5p, 219600, Qiagen). The dilutions were prepared in triplicate, using the same RT and PCR protocol as described above. The Ct values were plotted against the logarithm of the concentration, demonstrating a clear linear relationship between Ct value and Log (conc.). The resultant regression line was used to ascertain the concentration of miR-122 present in the samples. Acceptable repeatability was demonstrated by measuring the intra-assay variability of miR-122 duplicates (Karlen et al. , 2007) and expressed as concentration (fM) per MIQE guidelines (Bustinet al. , 2009), (CV: 0.25%, [0.11-0.49% IQR]). The intra-assay variation in miR-39 Ct values was assessed (CV median [IQR]: 3.04 [2.50-3.64] %). Reproducibility was determined by measuring inter-assay variability across plates and days by measuring miR-122 concentrations of reference samples. A no enzyme control (NEC), omitting the reverse transcriptase enzyme during reverse transcription, and no template control (NTC) omitting the cDNA in the RT-PCR plate were also included in every run. NEC and NTC controls had Ct values of >35. Ct values less than 35 were regarded as positive amplification signals.