Stratagene mx3000p instrument

Total RNA was extracted from the cerebrum and hippocampus using TRIzol reagents (15596-026, Life Technologies, Carlsbad, CA, USA). cDNA synthesis was performed using a reverse transcription kit (SG-cDNAS100; Smartgene, Daejeon, Republic of Korea). Quantitative PCR was carried out using SYBR Green (SG-SYBR-500, SmartGene, Ecublens, Switzerland) on a Stratagene Mx3000P instrument (Agilent Technologies, Santa Clara, CA, USA). The thermal protocol comprised three steps. In the first step, the temperature was maintained at 95 °C for 3 min. The second step involved 40 cycles, with each cycle comprising a denaturation step at 95 °C for 15 s, an annealing step at 60 °C for 15 s, and an extension step at 72 °C for 30 s. Finally, the third segment involved a melt curve analysis, involving a denaturation step at 95 °C for 10 s, an annealing step at 65 °C for 5 s, and a final extension step at 95 °C for 50 s. Transcript levels were determined by analyzing the cycle threshold and monitoring the amplification curve, with Rplp0 utilized as the internal control. The primer sequences can be found in Table 2.

The mRNA level of iNOS in lung tissue was determined by quantitative real-time polymerase chain reaction (qRT-PCR) as described previously (Rungsung et al. 2018 (link)). In brief, total RNAs were isolated from the collected lung tissues with the help of Trizol reagent (TAKARA, Beijing, China) and were finally dissolved in diethylpyrocarbonate (DEPC; MACKLIN, Shanghai, China)-treated ddH₂O. Afterwards, the first strand of cDNA synthesis was fulfilled using a BeyoRT™ III First Strand cDNA Synthesis Kit (Beyotime Biotechnology, Shanghai, China). Then, the iNOS expression was determined using BeyoFast™ SYBR Green One-Step qRT-PCR Kit (Beyotime Biotechnology) with a Stratagene Mx3000P instrument (Agilent Technologies, California, USA). GAPDH was used as the internal control, and the 2^-ΔΔCt method was used to calculate iNOS expression. The sequences were as follows: inducible nitric oxide synthase (iNOS) (Forward) 5′-GGTGCTATTCCCAGCCCAA-3′, (Reverse) 5′-AGTCACATGCAGCTTGTCCA-3′; GAPDH (Forward) 5′-AGACAGCCGCATCTTCTTGT-3′, (Reverse) 5′-CCGATACGGCCAAATCCGTT-3′.