Quantitative RT-PCR Analysis of Colon Tissue

The RNA samples for qPCR analysis were selected from the same colon tissues used for RNA sequencing. qPCR analysis was performed using a TB Green^® Premix Ex Taq™ II kit (Takara, Shanghai, China) and Bio-Rad C1000 Thermal Cycler Real-Time PCR System (Bio-Rad, Hercules, CA, USA). The reverse transcription reaction system is a final volume of 10 μL, including 1 μg RNA, 1 μL PrimeScript RT Enzyme MixⅠ, 1 μL RT Primer Mix, 4 μL 5 × PrimeScript Buffer and RNase-free water (37 °C for 15 min and then 85 °C for 5 s). Amplification volume was 20 μL containing 2 μL cDNA, 0.8 μL forward primer (10 μM), 0.8 μL reverse primer (10 μM), 0.4 μL ROX Reference Dye (50×), 10 μL SYBR Premix Ex Taq Ⅱ and 6 μL RNase free water. The amplification conditions were a pre-denaturation program (95 °C for 30 s), and the amplification program (95 °C for 5 s, and 60 °C for 34 s) was for 40 cycles. The expression level of Gapdh was normalized [12 (link)]. Table S1 provides detailed information on the primers used. The relative expression levels of gene expression were calculated by the ΔΔCt method.