Tranzol up reagent

Cells were extracted for total RNA with Tranzol UP reagent (Transgene), which were reversed transcribed with the Revert Aid First Strand cDNA Synthesis Kit (Transgene). The forward primer for BIRC2 gene was 5′-AGCACGATCTTGTCAGATTGG-3′ and corresponding reverse one was 5′-GGCGGGGAAAGTTGAATATGTA-3′. The primers for GAPDH were 5′-AGGGCTGCTTTTAACTCTGGT-3′ (forward) and 5′-TCTCGCTCCTGGAAGATGGTG-3′ (reverse). The primers of CFLAR were 5′-TCAAGGAGCAGGGACAAGTTA-3′ (forward) and 5′-GACAATGGGCATAGGGTGTTATC-3′ (reverse). PCR was performed with Fast Start Universal SYBR Green Master Mix (Roche, Basel, Switzerland), and the intensity of fluorescence was measured with the ABI 7500 Real-Time System (Applied Biosystems, Life Technologies). Each reaction was performed in triplicate in the condition as described previously [18 (link)]. GAPDH served as an internal control and the data were analyzed by the 2^−ΔΔC_t method.

Total RNA was extracted from frozen colon and ileum mucosal tissues (n = 3) using TranzolUp reagent (TransGen Biotech, China) according to the manufacturer's instructions. The concentration and quality of extracted total RNA were determined by a NanoDrop-ND2000 spectrophotometer (Thermo Fisher Scientific Inc., Germany). The integrity of total RNA was further checked by gel electrophoresis on a 1% agarose gel for visualization of complete 28 and 18S bands. Genomic DNA was eliminated by treatment with DNase I (TransGen Biotech, China). Complementary DNA (cDNA) was then synthesized from 1 μg of total RNA using an M-MLV First Strand Kit (Invitrogen, USA) following the instructions of the manufacturer. The qRT-PCR for gene expression was performed in duplicate using a ChamQTMSYBR®qPCR Master Mix (Vazyme Biotech Co., China) on a CFX connect system (Bio-Rad, USA). Specificity of the amplification was confirmed by the melting curve. Primers were designed using Primer Premier 5.0 software (Applied Biosystems, USA) and were synthesized by Generay Biotech Co. (Shanghai, China). Primer sequences, annealing temperatures (Tm), and product lengths of target genes are listed in Table S2. The fold change of the target genes was normalized to housekeeping gene (β-actin) and was calculated using the 2^−ΔΔCT method.