Maxime rt premix oligo dt15 primer

After total RNA extraction, single-stranded cDNA was generated using Maxime RT PreMix (Oligo dT₁₅ Primer; iNtRON Biotechnology, Kyungki-Do, Korea) following the manufacturer's directions. Synthesised cDNA fragments were amplified with the following primer pairs: ACTB, 5′-ACAACGGCTCCGGCATGTGCAA-3′ (sense) and 5′-CGGTTGGCCTTGGGGTTCAG-3′ (anti-sense); GAPDH, 5′-CTTTGGTATCGTGGAAGGA-3′ (sense) and 5′-CACCCTGTTGCTGTAGCC-3′ (anti-sense); NOX4, 5′-AGGAGAACCAGGAGATTGTTG-3′ (sense) and 5′-GGGATGACTTATGACCGAAAT-3′ (anti-sense); p22^phox, 5′-GTGTTTGTGTGCCTGCTGGAGT-3′ (sense) and 5′-CTGGGCGGCTGCTTGATGGT-3′ (anti-sense); EGFR, 5′-GCAAATAAACCGGACTGAAG-3′ (sense) and 5′-GTGGCACCAAAGCTGTATTTG-3′ (anti-sense); NOX2, 5′-GGGCTGTTCAATGCTTGTGGCT-3′ (sense) and 5′-ACATCTTTCTCCTCATCATGGTGC-3′ (anti-sense); NOX5, 5′-ATCAAGCGGCCCCCTTTTTTTCAC-3′ (sense) and 5′-CTCATTGTCACACTCCTCGACAGC-3′ (anti-sense). PCR was performed using a T-Gradient Thermal Cycler (Biometra, Goettingen, Germany) and AccuPower PCR Premix (Bioneer) according to the manufacturer's protocol. ACTB and GAPDH were used as the housekeeping genes. The PCR products were resolved on 1.5% agarose gels and visualised using a BioDoc-it Imaging System (UVP, Upland, CA, USA).

IL-8 as a proinflammatory cytokine marker gene was quantified using qRT-PCR. AGS cells (1 × 10⁵ cells/well) in a 6-well plate were co-cultured with 3-day-old H. pylori (MOI = 1:100). CLA or KPEVs-CLA at 0.01–0.12 µg/mL prepared in RPMI were added and incubated for 12 h. RPMI and KPEVs alone were used as untreated and vehicle controls. Cells were lysed and extracted using GENEzol (Geneaid Biotech, Taiwan) according to the manufacturer’s instructions. mRNA was converted into cDNA using Maxime RT PreMix Oligo(dT)15 Primer (iNtRON Biotechnology, South Korea). cDNA was amplified using SYBR iTaq DNA Polymerase reagent (Bio-Rad Laboratories, CA, USA) with a CFX96 real-time quantitative PCR system (Bio-Rad Laboratories, CA, USA). All primers were purchased from U2Bio, South Korea, and their sequences were as follows: IL-8-F, 5′-TCC AAA CCT TTC CAC CCC AA-3′; IL-8-R, 5′- ACT TCT CCA CAA CCC TCT GC-3′; GAPDH-F, 5′-CTG ACT TCA ACA GCG ACA CC-3′; GAPDH-F, 5′-GTG GTC CAG GGG TCT TAC TC-3′. The thermal cycling program was set as follows: pre-denature at 95°C for 5 min, denature at 95°C for 30s, annealing/extension at 58°C for 30s, and repeated for 40 cycles. The relative mRNA expression level was determined through the cycle threshold (Ct) normalized against GAPDH using the 2−ΔΔCt formula.