RT-qPCR Analysis of mRNA and miRNA

Initially, 1 μg of total RNA was used for the synthesis of the first strand of cDNA using PrimeScript 1st strand cDNA Kit (TaKaRa Bio, Kusatsu, Shiga, Japan) and 500 ng of total RNA for the synthesis of the first strand micro cDNA using Universal cDNA Synthesis kit II, 8–64 rxns (Exiqon, Vedbæk, Denmark), respectively. qRT-PCR was achieved using QuantiFast SYBR Green PCR kit (QIAGEN, Hilden, Germany). The relative expression level of mRNAs and miRNAs were calculated using the 2^-ΔCt method [45 (link)]. GAPDH (NCBI accession no. 2597) and β-ACTIN (NCBI accession no. 60), and miR-191-5p (miRBase accession no. MIMAT0000440) and U6 snRNP (NCBI accession no. 26827) were used as endogenous controls for normalizing of mRNA and miRNA expression levels, respectively. GAPDH and β-ACTIN were already reported to be used for the neurological studies [46 (link)], U6 is a universal reference for miRNA RT-qPCR studies [47 (link)], and miR-191-5p was also reported to be used for qRT-PCR of blood samples [48 ] and was introduced as a reliable endogenous control by the Exiqon company (miRCURY LNA Universal RT microRNA PCR system). Normalization was performed using geometric averaging [49 (link)] of miR-191-5p and U6 snRNP, GAPDH, and β-ACTIN expressions.