Exicycler real time quantitative thermal block

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).

After five days of treatment, total RNA from wild-type worms was extracted using the Trizol method (Invitrogen, Carlsbad, CA, USA). 1000 ng of total RNA was taken for reverse transcription using Accupower RT Premix (Bioneer, Korea) for first-strand cDNA synthesis, following the manufacturer's protocol. The quantitative real-time (qPCR) analysis was carried out on an Exicycler Real-Time Quantitative Thermal Block (Bioneer) with SYBR green and Green Star PCR Master Mix (Bioneer). The relative gene expression level was calculated using the 2^-ΔΔCt method^{76 (link)}, keeping act-2 as a housekeeping gene to normalize the gene's expression data. Primers used in this assay are mentioned in Supplementary Table S1 and the gene descriptions are mentioned in Supplementary Table S2.