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Quantitative RT-PCR analysis

TY Tao Yang
XQ Xiaoye Qu
JZ Jiaying Zhao
XW Xiao Wang
QW Qian Wang
JD Jingjing Dai
CZ Chuanlong Zhu
JL Jun Li
LJ Longfeng Jiang
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The RT-PCR was performed as described [26]. Total RNA was purified from liver tissue or cell cultures using RNeasy Mini Kit (Qiagen, Chatsworth, CA) according to the manufacturer’s instructions. The total RNA (2.5 μg) was reverse-transcribed into cDNA using the SuperScript™ III System (Invitrogen, CA). Quantitative real-time PCR was carried out using the QuantStudio 3 (Applied Biosystems by ThermoFisher Scientific). The following were added in the final reaction volume of 25 μl: 1 × SuperMix (Platinum SYBR Green qPCR Kit; Invitrogen) cDNA and 10 μM of each primer. The amplification conditions were 50 °C (2 min), 95 °C (5 min), followed by 40 cycles of 95 °C (15 s) and 60 °C (30 s). Primer sequences used to amplify Tnf-α, IL-1β, IL-6, Mcp1, Cxcl-1, Inf-β, Nqo1, Gclc, and Gclm are shown in Table S1. Target gene expressions were calculated by their ratios to the housekeeping gene β-actin.

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