Miscript mirna assays

RNA was extracted from plasma (331 patients, OMEGA-REMODEL trial) using miRCURY RNA Isolation Kit – Biofluids (Exiqon) and reverse transcribed using miScript II RT Kit (Qiagen, Fredrick, MD, USA). cDNA samples were pre-amplified with miScript Microfluidics PreAMP Kit (Qiagen, Fredrick, MD, USA) and RT-qPCR miRNA profiling analyses performed on the BioMark System (Fluidigm Corp. South San Francisco, CA) using miScript miRNA Assays (Qiagen, Fredrick, MD, USA) and Dynamic Array 96.96 (Fluidigm Corp. South San Francisco, CA). A Ct value of 23 was set as the cut-off point for detectable miRNA species. Measurement of RNAs on the Fluidigm platform has previously been extensively validated against conventional RT-qPCR (Jang et al., 2011 ). The qPCR raw data has been deposited in GEO (accession number: GSE112690).

Total RNA extracted from plasma samples via High Pure miRNA Isolation Kit (Roche Life Science, Mannheim, Germany). Total RNA was reversely transcribed to cDNA by using miScript II RT Kit (Qiagen, Hilden, Germany). Obtained cDNA samples are PreAmplified via miScript Microfluidics PreAMP Kit (Qiagen, Hilden, Germany). Quantitative real-time polymerase chain reaction analysis was carried out using miScript miRNA Assays (Qiagen, Hilden, Germany) with Dynamic Array (Fluidigm, South San Francisco, CA, USA) on BioMark System (Fluidigm, South San Francisco, CA, USA) as per manufacturer's protocol. The relative gene expression was calculated with the comparison of cycle times for target polymerase chain reaction using this equation: relative gene expression = 2^{−(ΔCtsample − ΔCtcontrol)}.