Total RNA was isolated from plasma using the MirVana PARIS microRNA isolation kit (Ambion/Applied Biosystems), according to the manufacturer's instructions.
Label
SYBR Green
Label protocol
Global profiling of miRNAs in the plasma samples was carried out using the BioTrove OpenArray platform (Applied Biosystems), according to the manufacturer’s instructions. For each sample, two RT reactions were performed using MegaPlex RT primer pools A and B (Applied Biosystems) on a LabNet Multigene Gradient with 40 cycles of 2 minutes at 16°C, 1 minute at 42°C, and 1 second at 50°C, followed by 5 minutes at 85°C. The entire RT reaction was used for preamplification. Preamplification was carried out on a ViiA 7 instrument (Applied Biosystems) using MegaPlex RT preamplification primer Pools A and B and MegaPlex preamp Master Mix (Applied Biosystems) under the following conditions: 10 minutes at 95°C, 2 minutes at 55°C, 2 minutes at 72°C, followed by 16 cycles of 15 seconds at 95°C, and 4 minutes at 60°C. Preamplification reactions were then incubated at 99.9°C for 10 minutes and cooled to 4°C. The resultant cDNA was diluted, and this frozen at -20°C until further use. The diluted cDNA was thawed, combined with the OpenArray real-time PCR Master Mix and loaded onto the OpenArray miRNA panel plates (Applied Biosystems) using the AccuFill autoloader. The loaded plates were run on the BioTrove OpenArray realtime PCR instrument (Flinders Medical Centre, SA) and run according to the default protocol for reaction conditions.
Hybridization protocol
n/a
Scan protocol
n/a
Description
test 5048.1
Data processing
MiRNAs that are missing in > 50% of samples were excluded to acquire acceptable distribution of non-detects for down-stream analysis. Quantile normalization was used to adjust for technical variability across multiple samples. Missing data was imputed using the nearest-neighbour method (KNNimpute). The pre-processing of miRNA cycle quantification (Cq) values from quantitative RT-qPCR assays were performed using MATLAB 2014b, Bioinformatics Toolbox and Statistics Toolbox.
