2.2. RT-PCR

SuperScript™ IV One-Step RT-PCR System (Invitrogen, Thermo Fisher Scientific, and USA) was performed to evaluate the quantification of mRNA according to the manual and experimental protocol. The primers used for RT-PCR assay were as follows: CKIP-1, forward 5′-AATTCTGCGGGAAAGGGATTT-3′, reverse 5′-AACACCTCCTGACTGTTTTTCTC-3′ and β-actin, forward 5′-CTCCATCCTGGCCTCGCTGT-3′, and reverse 5′-GCTGCTACCTTCACCGTTCC-3′. The program thermal cycler at 60°C 10 min and 98°C 2 min for 1 cycle; 98°C 10s, 55°C 10 s, and 72°C 30 s for 40 cycles; and 72°C 5 min for 1 cycle. The relative quantification of mRNA levels was determined using the 2^−ΔΔCt method after being normalized to β-actin.