Quantification of Gene Expression Pathways

Gene expression for the TLR pathway including TLR2, TLR4, MYD88, and NFκB; for the Nrf2 pathway, including Nrf2, HO-1, and NQO-1; and for apoptotic markers, including Bax and Bcl2 were quantified via real-time PCR (qPCR), consuming the primers’ sequences in compliance with the method described elsewhere [30 (link)]. Briefly, RNA was isolated and purified using the Trizol reagent kit (Invitrogen, Carlsbad, CA, USA), then reverse transcribed using the reverse transcription-polymerase chain reaction (RT-PCR) kit (TaKaRa, Kusatsu, Shiga, Japan), following the manufacturer’s procedures. qPCR was applied using SYBR ExScript RT-PCR kit, and quantification examinations were completed via Opticon-2 Real-time PCR reactor (MJ Research, Capital Court, Reno, NV, USA). qPCR results were obtained using Step PE Applied Biosystems (Waltham, MA, USA) software. Target gene expressions were evaluated and correlated to the β-actin as the reference gene, and the results are shown in figures as relative expressions. The primer sequences used in this study were mentioned in Table 1.