Quantitative Analysis of miRNA and mRNA

RNA extraction and RT-qPCR were performed as described [12] (link). Total RNA was extracted using Tri Reagent (Sigma-Aldrich Co., St. Louis, USA) according to the manufacturer's instructions. For miRNA qPCR, the expression levels of specific miRNAs were detected using stem-loop RT-PCR [21] (link). The universal PCR reverse primer for the miRNAs was 5′-GTGCAGGGTCCGAGGT-3′. Amplification of marker genes was performed using specific primers, Maxima SYBR Green qPCR Master Mix (K0222, Fermentas) and a StepOne sequence detector (Applied Biosystems, USA). Designed primers for each miRNAs are listed in Table S1; endogenous U6 and miR-16 were used as an internal control for normalizing miRNA expression in cells and plasma respectively [12] (link). The VEGF mRNA expression data were normalized to the average value of GAPDH and beta-actin.