Quantifying miRNA Expression by RT-qPCR

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)].