Real-time qPCR analysis of CYP3A4 and MDR1 mRNA in LS174T

To determine CYP3A4 and MDR1 mRNA induction, LS174T cells were transfected with 100 ng/well PXR expression plasmids or pcDNA3.1 vector or weren’t transfected. Following 24 h transfection, appropriate cell samples were exposed to phenobarbital (1 mM), SGD (1 mg/ml), SY (1 mg/ml), GC (1 mg/ml) or the solvent control (0.1% DMSO) for 48 h, respectively. Then cells were harvested and the mRNA levels of CYP3A4, MDR1 were measured by real-time qPCR.

Total RNA isolation was performed using TRIzol reagent (Invitrogen) and converted into cDNA using the Fermentas RT kit according to the manufacturer’s instructions. The primers designed using Primer Premier 5.0 are presented in Table 1. PCR was performed in a total reaction volume of 30 μL, containing 15 μL 2 × SYBR Green PCR Master Mix (Applied Biosystems), 3 μL cDNA, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), and 10 μL double-distilled water. Real-Time qPCR analysis was performed using the Applied Biosystems 7500 real-time PCR system. The temperature profile was 95 °C for 10 min; 40 cycles of 95 °C for 10 s, 59 °C for 50 s; melting curve program 60–95 °C. Real-time qPCR for each gene of interest was performed in triplicate, and the expression values were calculated as their average. Gene expression was evaluated in at least three independent experiments and each performed in triplicates. The expression of the target genes were normalized to the reference gene β-actin and then processed using the 2^−ΔΔCt method [31]. The data are expressed as the fold changes in the activation of gene expression relative to the non-transfected group with 0.1% DMSO treatment (set to be 1).

Primer sequences used for real-time qPCR