Power taq pcr mastermix

The treated cells were collected by groups. TRIpure extraction kit (RP1001; BioTek, Beijing, China) was used as instructed to extract the total RNA in each group. The instrument NanoDrop-2000 (Thermo Fisher Scientific, MA, USA) was used to determine the RNA concentration in each group. Real-time polymerase chain reaction (PCR) (Exicycler 96, BIONEER, Daejeon, Republic of Korea) was used, and super M-MLV reverse transcriptase (PR6502; BioTek) was used for reverse transcription. 2× Power Taq PCR MasterMix (PR1702; BioTek) and SYBR Green I (SY1020; Solarbio) were used for PCR. β-actin was used as a reference. The formula 2^−ΔΔCt was used to calculate the relative expression quantity of mRNA of TNF-α gene. See Table 1 for primer sequence. It is synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China).

Total RNAs in ischemic penumbra part of brain tissues was extracted using the RNA Purified Total RNA Extraction Kit (RP1201, BioTek, Beijing, China). cDNA templates were achieved by reversely transcribing the RNA using Super M-MLV reverse transcriptase (RP6502, BioTek, Beijing, China). The reaction mixture contained 10 μl of 2×Power Taq PCR MasterMix (PR1702, BioTek, Beijing, China), 0.5 μl of each primer (IL-1β, forward: 5′-GCAATGGTCGGGACATAGTT-3′, backward: 5′-CAGAGGCAGGG AGGGAAA-3′. IL-6, forward: 5′-AACTCCATCTGCCCTTCA-3′, backward: 5′-CTGTTGTGGGTGG TATCCTC-3′. TNF-α, forward: 5′-TGGCGTGTTCATCCGTTCT-3′, backward: 5′-CCACTACTTCAGCGTCTCGT-3′. β-actin, forward: 5′-GGAGATTACTGCCCTGGCTCCTAGC-3′, backward: 5′-GGCCGGACTCATCGTACTCCTGCTT-3′), 1 μl of the cDNA template, and 8 μl of RNase-free H₂O. Amplification was performed following a denaturation step at 95°C for 10 min, and then 40 cycles at 95°C for 10 s, 60°C for 20 s, and 72°C for 30 s, and stopped by 25°C for 5 min. Relative expression levels were calculated with ExicyclerTM 96 (BIONEER, Daejeon, Republic Korea) according to the expression of 2^−ΔΔct.

Real-time fluorescence quantitative PCR was used to detect the relative expression of the Bax and caspase-3 genes in each group of cells. According to the trizol extraction kit (15596026; Thermo Fisher Scientific, Waltham, MA, USA) experimental instructions, total RNA was extracted from the cells, and the RNA concentration was detected using a NanoDrop 2000 (Thermo Fisher Scientific). An Invitrogen reverse transcription kit (K1691; Thermo Fisher) was used for reverse transcription. Different primers were designed based on the Bax and the caspase-3 genes. The primer sequences are shown in Table 1 and were synthesized by Biotechnology Co., Ltd. (Shanghai, China). PCR using 2 × Power Taq PCR mastermix (PR1702; Biotek, Beijing, China) and SYBR Green I (SY1020; Solarbio, Beijing, China) was performed on a fixed light quantifier. The relative expression of these two genes was calculated by the formula 2_−ΔΔCT.