Real-time quantitative PCR analysis of NF-kB, TNFR1, BRAF, MEK and NRF2 genes in human D24, MM418-C1 melanoma and HaCaT cells

D24, MM418-C1 melanoma and HaCaT cells were treated with 100 µg/mL of the different M. cochinchinensis seed extracts for 48 h prior to RNA collection. Results were presented as the fold change compared to untreated controls ± SEM (n=3), different letters indicated statistical significance at p≤0.05 between different treatments (Analysis by One-way ANOVA with Turkey’s LSD test). RNA was extracted using TRIzol™ Reagent (Life Technologies, Melbourne, Australia) as per the manufacturer’s protocol. RNA was quantified spectrophotometrically at 260 nm (Nanodrop ND1000), and its purity checked using the ratios of absorbance at 260/280 nm and 260/230 nm. cDNA was synthesized from 1 μg total RNA using SuperScript III reverse transcriptase (Thermo Fisher Scientific) with oligo (dT) primers according to the manufacturer’s instructions. Each transcript quantification was carried out in triplicate. Subsequently, 4 µL (10 ng/µL) cDNA was added to 10 µL SYBR mix, 0.8 µL of each (10 µM) forward and reverse primer, and 4.4 µL DEPC H2O. The qPCR was performed on a Qiagen Rotor-Gene Q (Qiagen, Germany) and analysed with inbuilt software (version 2.3.1.49). The thermal cycling conditions were as follows: initial activation at 95°C for 2 min, 39 cycles of 95°C 5 s, 60°C for 10 s, 72°C for 15 s, and melting curve from 65°C to 95°C. Relative quantitative analysis of TNFR1, NF-kB, BRAFWT, BRAFV600E and Nrf2 mRNAs were performed using the 2−ΔΔCq method which was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The expression was calculated as n-fold induction of the gene of interest in treated cells relative to that of untreated control cells