Green star pcr master mix

The TRIzol kit (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA from wild-type nematodes treated with different concentrations of C. nervosum fruit extracts (20 and 30 μg/ml). From the total RNA, 1000 ng was converted to cDNA using AccuPower RT Premix (Bioneer, Korea) with oligo dT primers following the manufacturer's protocol. Real-time PCR was carried out using SYBR Green, Green Star PCR Master Mix (Bioneer, Korea), in the Exicycler Real-Time Quantitative Thermal Block (Bioneer, Daedeok-gu, Korea) with the help of gene-specific primers. The expression data were normalized to the internal control actin and then represented as upregulated or downregulated by normalizing with the untreated control. The sequences of the primers are given in Table 1.

In brief, total RNA was isolated from specific treatment cells using Trizol reagent). Using Accupower RT Premix (Bioneer), 1 μg of total RNA was converted to cDNA. Quantitative real-time PCR reaction was performed by using the Green Star PCR Master Mix where SYBR Green was included (Bioneer). Then, the specific genes CAT, SOD1, SOD2 and GPx were determined by the Exicycler Real Time Quantitative Thermal Block (Bioneer). The specific primers were previously reported by our research group [19 (link)]. The relative expression of each gene was normalized to the internal control gene (β-actin).

Total RNA was extracted from HaCaT-TNF-α cell lines using Genezol reagent (GeneAid, Taiwan). cDNA was synthesized from 1 μg of total RNA using Accupower RT Premix (Bioneer, Korea) with oligo (dT) 18 primer following the manufacturer's protocol. Quantitative real-time PCR for IL-1β, IL-6, IL-8, NF-κB1, KRT16, FOSL1, MMP9 and GAPDH was performed by using Green Star PCR Master Mix (Bioneer, Korea) and gene-specific primers, as listed in Table 1, following the manufacturer’s instructions. The PCR for all genes were performed and fluorescent signals were measured in real time under the following conditions: 95°C for 10 min, 35 cycles at 95°C for 15 sec, 54°C for KRT16, 55°C for FOSL-1 and IL-1β, 57°C for GAPDH and MMP9, 58°C for IL-8, NF-κB1 and IL-6 for 30 sec. The fluorescent signal was expressed as the number of cycles required to achieve fluorescence at threshold. To normalize the amount of total RNA added to each reaction, GAPDH was used as an internal control and relative gene expressions were presented with the 2^-ΔΔCt method [37 (link)].