Iq5 real time quantitative pcr

Total RNA of cells was extracted using RNA isolation reagent (Invitrogen, USA) and then was reverse-transcribed using One Step RT-qPCR Kit (Sangon Biotech, China) according to the manufacturer's protocol. Next, equal amounts of cDNA were used for RT-qPCR. PCR reaction and real-time detection were performed using iQ5 Real-time Quantitative PCR (Bio-Rad, USA). As shown in Table 1, the appropriate forward and reverse real-time PCR primers were used for GAPDH, VEGF, Bcl-2, BAX, LC3B, Caspase 3, mTOR, and Beclin-1. The real-time PCR cycles included predenaturation at 95°C for 5 min, 40 cycles of denaturation at 95°C for 20 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. 2^−ΔΔCt (ΔΔCt = (C_mRNA–Ct_GAPDH) − (control − Ct_GAPDH)) was used to quantify the relative expression of target mRNA.

After treatment, HBMVECs were collected, of which total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. Total RNA of infarct tissue of the brain was extracted similar to HBMVECs. Total RNA was reverse-transcribed using the One-Step RT-qPCR Kit (Sangon Biotech, China) and then amplified with PCR primers on the iQ5 Real-Time Quantitative PCR (BioRad, USA). Real-time PCR primers for Caveolin-1, mTOR, BCL-2, Beclin-1, Caspase-3, and VEGF are shown in Table 1. The PCR cycling profile included predenaturing at 95°C for 3 min, 40 cycles of denaturing at 95°C for 15 sec, annealing at 60°C for 20 sec, and extension at 72°C for 30 sec. The 2^−ΔΔCt method (ΔΔCt = (C_{target mRNA} − Ct_ACTB) − (control − Ct_ACTB)) was used to quantify the relative expression of the target mRNA. Relative expression of mRNA was normalized to the control. ACTB was chosen as the internal control.