Quantifying Gene Expression by qRT-PCR

The relative mRNA levels were assessed by quantitative reverse transcription PCR (qRT-PCR) [38 (link)]. After Trizol (Invitrogen, Carlsbad, CA) treatment, all RNAs of the liver tissues were obtained and the Prime Script Kit (TaKaRa Bio Inc., Japan) was performed for cDNA synthesis. qRT-PCR was performed on a ViiATM7 RT-PCR system (Applied Biosystems, Carlsbad, CA) using SYBR Green fluorescent-based assay (638320, TaKaRa Bio Inc. Japan). The β-actin gene was used as an internal standard and after normalizing to its expression level, the relative expression levels of α4nAChR, α7nAChR, and α-SMA were quantified by the 2^−ΔΔCt method [39 (link)]. The reaction parameters were set as 50°C for 2 min, 95°C for 15 s, 95°C for 15 s, and 60°C for 1 min for 40 cycles. The primers of target genes were synthesized by Sinaclon (Tehran, Iran) and reported in Table 1.