2.7. Determination of Pro-Inflammatory Cytokine Gene Expression by Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

The mRNA was extracted from defrosted liver and colonic epithelium using Purezol reagent according to the instructions presented in the user manual. Accordingly, mRNA was then synthesized to cDNA using a high-capacity cDNA reverse transcription kit, according to the manufacturer’s instructions. The qPCR amplification was carried out in the QuantStudioTM 6 Flex System (Thermo Fisher Scientific, Waltham, MA, USA) using a SensiFAST SYBR Lo-ROX Kit at 95 °C for 2 min, followed by 40 cycles at 95 °C for 5 s, 60 °C for 10 s, and 72 °C for 20 s. Gene expression was standardized to β-actin levels and measured using the 2^−∆∆ct technique [29]. Table 1 presents the primer lists [29].

Primer sequences of qRT-PCR.