Determination of SOD1, GPx1, CAT, Nrf-2, ICAM-1 and Akt1 mRNA levels using quantitative Real-Time PCR (q-RT-PCR) technique

Total RNA was extracted from rat embryos and hepatic tissues of rat fetuses by using the Quick-RNA TM MiniPrep Plus Kit (ZYMO RESEARCH, catalog nos. R1057 and R1058). RNA samples were transcribed into cDNA using the iScript™ cDNA Synthesis Kit (Bio-RAD, USA, catalog number: 1708891). Specific primers purchased from Macrogen, Korea (shown in Table ​Table1)1) were used for RT-PCR to determine the levels of the steady-state mRNA of desired proteins (SOD1, GPx1, CAT, Nrf-2, ICAM-1, and Akt1). The iTaq™ Universal SYBR ® Green Supermix (Bio-Rad, USA, catalog number: MLL4801) was used in this quantitative detection. Real-time PCR reactions were performed in triplicates by using a thermal cycler with a CFX Connect Real-Time PCR Detection System (BIO-RAD, USA, catalog number: 1855200). The comparative CT method which depends on the value of the studied genes to the reference gene was used to calculate the fold difference in gene expression (2^−ΔΔCt ) with beta-actin (β-Actin) and TATA box binding protein (TBP) as reference genes and control untreated samples as calibrator (2^−ΔΔCt = 1).

Specific forward and reverse primers (From macrogen) designed for oxidative stress, inflammation and apoptosis-related genes (SOD1, GPx1, CAT, Nrf-2, ICAM-1 and Akt1), and for reference endogenous genes (ß- Actin and TBP) in rat liver.