2.6. Quantitative Real-Time PCR

Total RNA (1 μg) isolated from HepG2 cells treated with DMSO (control), E2, PPT, or DPN (n = 3 for each treatment group) was reverse transcribed into cDNA using a cDNA synthesis kit (Bio-Rad, Hercules, CA). The primers were synthesized by Integrated DNA Technologies (San Jose, CA). Relative expression of three differentially expressed genes indicated by RNA-Seq and known to be regulated by estrogens, PPARG, SOCS3, and IL6R, was measured in this preliminary study. Other identified differentially expressed genes will be validated in the future. ACTB was used as a reference gene, since ACTB mRNA level did not vary among groups with different treatments according to RNA-Seq analysis. ACTB forward primer was GTG GGG CGC CCC AGG CAC CA, and reverse primer was GTC CTT AAT GTC ACG CAC GAT TTC. PPARG forward primer was TCT GGC CCA ACT TTG GG, and reverse primer was CTT CAC AAG CAT GAA CTC CA. SOCS3 forward primer was GGA GTT CCT GGA CCA GTA CG, and reverse primer was TTC TTG TGC TTG TGC CAT GT. IL6R forward primer was CAG CTG AGA ACG AGG TGT CC, and reverse primer was GCA GCT TCC ACG TCT TGA. Quantitative real-time PCR was carried out using SYBR green master mixes and an iCycler (Bio-Rad, Hercules, CA). Amplified products were confirmed via gel electrophoresis and melt curve analysis. Results were generated from triplicate experiments. Relative quantification of gene expression was normalized using the housekeeping gene ACTB, calculated by the 2^−ΔΔCt method [43], and presented using the control group as 100%.