Oligo dt and random primers

Cell viability was assessed according to a previously described method^{50 (link)}. Chicken embryo primary hepatocytes were seeded at a density of 10000 cells/well in 96-well plates and cultured overnight. The hepatocytes were treated with different concentrations of AFB₁ (0–10 μg/mL), ZEN (0–120 μg/mL), or OTA(0–10 μg/mL). After 48 h, 0.5 mg/mL MTT (Sigma-Aldrich, Shanghai) was added to each well. We extracted the total RNA from mycotoxin-treated or untreated chicken primary hepatocytes using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA (2 μg) was reverse transcribed using M-MLV reverse transcriptase (Promega, Madison, WI, USA) with oligo d(T) and random primers (Takara Biotechnology, Dalian, China). The obtained cDNA products were used as templates for CYP1A4, CYP2D20 and CYP3A37 transcript quantification by RT-PCR. GAPDH was used as an internal reference to normalize gene expression. The expression levels were calculated using the 2−ΔΔCt method^{51 (link)}.

Hepatic RNA from three groups (n = 10 in each group) was extracted using an RNeasy Mini Kit (Qiagen, Germantown, MD, United States), and cDNA was synthesized using oligo-dT and random primers (TaKaRa, Shiga, Japan). Quantitative real time-PCR (qPCR) was performed using a SYBR green real-time PCR master mix (Applied Biosystems, Foster City, CA, United States) on the ABI 7900 detection system (Applied Biosystems, Foster City, CA, United States). Gapdh was used as the internal control. Primers are listed in Table 2.

Placental RNA was extracted using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol. DNA contamination was wiped out by DNase I digestion (AM2222, Thermo Fisher Scientific). Total RNA was reverse-transcribed from 1 μg of RNA using oligo dT and random primers (TaKaRa, Japan) and amplified by SYBR® Premix Ex Taq™ (TaKaRa, Japan) in the ABI Prism 7900HT (Applied Biosystems, Foster City, CA). All threshold cycle (Ct) values of each sample were utilized in the post-PCR data analysis. GAPDH is our internal control, and gene expression levels were normalized against GAPDH. Full list of primer sequences is shown in Supplemental .