Super m mlv reverse transcriptase

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.