Plasma miRNA profiling using OpenArray

Small RNAs were isolated from 50 µL of plasma using Serum/Plasma small RNA kit (Qiagen, West Sussex, UK) to produce small RNA suspended in RNase free water. MiRNA profiling was performed using the OpenArray platform (ThermoFisher Scientific, Waltham, MA, USA) [27 (link),28 (link)]. OpenArray reverse transcription reaction was performed according to the manufacturer’s protocol using small RNA pooled from 10 samples. cDNA was pre-amplified and loaded onto the OpenArray automatically by the OpenArray AccuFill System (ThermoFisher Scientific) and run on a QuantStudio 12K Flex Real-Time PCR system. A total of 754 human miRNAs were amplified from each sample together with 16 replicates of 4 internal controls (ath-miR159a (negative control), RNU48, RNU44, and U6 rRNA). OpenArray Ct values were normalised to the global mean (GMN) [27 (link)].