Cardiovascular Disease miRNA Profiling

Sequences of 84 different predesigned mature miRNAs (listed in Supplementary Table S3) were detected using a Human Cardiovascular Disease miScript miRNA PCR Array (MIHS-113Z, Qiagen, Hilden, Germany), as previously described, containing a miRNA sequence from C. elegans as an exogenous normalizer (spike-in cel-miR-39) [36 (link)]. All cDNA steps and PCR setup were performed via a QuantStudio™ 5 Flex Real-Time PCR System (Applied-Biosystems, Carlsbad, CA, USA). The PCR cycling was performed according to the manufacturer’s protocol. Briefly, only miRNAs with Ct values < 30 in all samples were considered. miRNA normalized expressions were represented by ∆Ct, calculated by subtracting the global geometric mean signal from individual miRNA Ct values. The 2−∆∆Ct method was used to calculate miRNAs’ fold change.