2.13. Quantitative Reverse Transcription Polymerase Chain Reaction Analysis of Antioxidant Enzymes Gene Expression

U937 monocyte cells were seeded in 6-well plates at 7 × 10⁵ cells per well in a final volume of 3 mL/well of RPMI with 10% FBS, 100 U·mL⁻¹ Penicillin, 100 μg·mL⁻¹ Streptomycin and 1 mM Na Pyruvate for 30 h at 37 °C. The cells were then supplemented with extracts from 3-day-germinated mung bean as treatment, or ethanol as control, for 24 h. The medium including extract and ethanol was removed by collecting the cells and then centrifuging them for 5 min at 300× g. Subsequently, cells were resuspended in RPMI with 10% FBS, 100 U·mL⁻¹ Penicillin, 100 μg·mL⁻¹ and 1% Sodium Pyruvate and returned to the 6-well plates. The resuspended U937 cells were treated with 1 mM PQ and 0.7 mM SNAP, or with an equal amount of RPMI and PBS for the control and incubated for 16 h. Using TRI Reagent^® Solution (Invitrogen, Oxford, UK), total RNA extraction was performed following the manufacturer’s instructions. The ratio A260/A280 was undertaken to check the RNA purity, utilizing a UV/VIS spectrophotometer (ThermoSpectronic, Helios γ, Waltham, US). Reverse Transcription-PCR was performed with 100 ng RNA using Super Script III Reverse Transcriptase (Invitrogen, Oxford, UK) and random hexamers as primers (Promega, Southampton, UK) to obtain cDNA. A quantitative real time PCR (qPCR) reaction was then performed on the obtained cDNA with SYBR Green PCR Master Mix kit (Primer design, Camberley, UK), as recommended by the manufacturer. Each reaction was done in duplicate accompanied by the relevant negative control. The sequences of oligonucleotide primers for qPCR (Table 1) were designed by Doctors Bermano and Goua based on previously published data [32,33], and passed the required efficiency tests. The comparative Ct method was used to analyze the mRNA levels. The target gene expression was related to the expression of β2 microglobulin (β2M), the house keeping gene. Experiments were carried out 3 times independently with duplicates for each treatment in each experiment.

Primers sequences.

F—forward primer, R—reverse primer; GPx Glutathione Peroxidase, SOD Superoxide Dismutase.