Mx3000p real time pcr

VSMCs from the control group, the LV-NELIN-siRNA-VSMCs group and the NELIN-VSMCs group were selected, respectively. Total RNA was isolated from the cultured cells using TRIzol and reversely transcribed to cDNA using the PrimeScript^® 1st Strand cDNA Synthesis kit according to the manufacturer's instructions. PCR was performed on an Mx3000P real-time PCR instrument (Agilent Stratagene, Amsterdam, Netherlands) using the manufacturer's instructions of the SYBR^® Premix Ex Taq™ kit. The primers of target genes were designed and supplied by Invitrogen Life Technologies and shown in Table I. Each RT-qPCR reaction mix consisted of 12.5 µl SYBR^® Premix Ex Taq™, 3 µl cDNA, 0.2 µL each primer (10 µM) and 9.1 µl PCR-grade water, in a final volume of 25 µl. Thermal cycling conditions included an initial step at 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and 60°C for 30 sec. The final extension step included 40 cycles at 95°C for 1 min, 60°C for 30 sec and 95°C for 30 sec. Data were calculated by the ^ΔΔCt method and compared to a reference (GAPDH). All experiments were performed in triplicate and repeated in at least three separate experiments.

RNA extracted from tissue samples were isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. The primer sequences for qPCR are displayed in Table S1. qPCR analyses were performed on MX3000p real-time PCR (Agilent, USA). The relative miRNA and mRNA expression levels were analyzed using the 2^–ΔΔCt method.