Total RNA was extracted using NucleoSpin RNA Plus XS kit, and 1 μg total RNA was used for cDNA synthesis by PrimeScript RT-PCR kit (TaKaRa, Dalian, China). The mRNA levels were examined using SYBR Green Supermix kit (Bio-Rad). All these procedures were performed according to the manufacturer's instructions. The qPCR was performed using the CFX96 Touch Real-Time PCR Detection System under conditions: 30 seconds initial denaturation at 95°C, then 40 cycles of 10 seconds at 95°C, and 30 seconds at 60°C. AKAP-9 mRNA levels were normalized to GAPDH levels. The primers were adopted from previous study [24 (link)] and listed below.
AKAP9-forward: 5′-ACTCAAGGCACAGCATAAACAC-3′
AKAP9-reverse: 5′-GTTCTTCACTGCGTC CCAA-3′
GAPDH-forward: 5′-ACAGTCAGCCGCATCTTCTT-3′
GAPDH-reverse: 5′-GACAAGCTT CCCGTTCTCAG-3′
AKAP9-forward: 5′-ACTCAAGGCACAGCATAAACAC-3′
AKAP9-reverse: 5′-GTTCTTCACTGCGTC CCAA-3′
GAPDH-forward: 5′-ACAGTCAGCCGCATCTTCTT-3′
GAPDH-reverse: 5′-GACAAGCTT CCCGTTCTCAG-3′
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