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.