Mirna specific miscript primer assay

RT-qPCR reactions were performed and reported according to the MIQE guidelines [45 (link)]. For quantification of individual miRNA expressions, cDNA was synthesized from 500 ng total RNA with 4 μl of HiSpec Buffer, 2 μl of Nucleics Mix and 2 μl miScript RT Mix (miScript II RT Kit, Qiagen; 218161) in a final volume of 20 μl. This reaction mix was incubated for 60′ at 37°C and 5′ at 95°C using an iCycer instrument (Bio-Rad). qPCR reactions contained 3 ng of cDNA, 2.5 μl QuantiTeckt Mastermix, 0.5 μl miScript Universal Primer and 0.5 μl miRNA-specific miScript Primer Assay (Qiagen, miScript Primer Assays used are listed in Supplementary Table S2) in a total volume of 5 μl. Expression levels were normalized against three stably expressed reference miRNAs (hsa-miR-125a, hsa-miR-423 and hsa-miR-92) validated with GeNorm [46 ] and analyzed using qbase+ software version 2.6 (http://www.biogazelle.com/qbaseplus) [47 (link)].

For the hsa-miR-200c-3p expression study, we applied previously described experimental protocols [23 (link)]. The expression level of hsa-miR-200c-3p was analyzed on all recruited patients (24 CTR subjects and 72 CAD patients) by qRT-PCR using a miScript SYBR Green PCR kit (QIAGEN, https://www.qiagen.com/us, accessed on 31 December 2022) and a miRNA-specific miScript primer assay (QIAGEN). RNA U6 was used for data normalization and analysis of the results since it showed stable expression among our samples. Data analysis was performed using the comparative threshold cycle (Ct) method quantification (2-ΔCT method) (PROTOCOL DOI: dx.doi.org/10.17504/protocols.io.zp7f5rn; accessed on 31 December 2022).