Quantifying miRNA and mRNA Expression

Analyses of miR-431, Dkk1, Krm1, and Wnt in the brain were performed according to routine lab protocols (Wu et al., 2012 (link)). Real-time PCR reactions were carried out using EXPRESS SYBER® GreenER™ qPCR SuperMix Universal (Thermo Fisher Scientific) in triplicates for each cDNA sample on Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific). Primers specific for each miRNA and mRNA were obtained using Invitrogen (Thermo Fisher Scientific). As an internal control, primers for S12 were added for RNA template normalization, and the relative quantification of gene and miRNA expressions was calculated against S12 using a 2-ΔΔCT method. Gene-specific primer for miR-431: 5′-CAGGCCGTCATGCAAA-3′.