Rna isolation reagent

Total RNA was isolated from liver samples using the RNA Isolation Reagent (Vazyme Biotech Co., Ltd., Nanjing, China). The concentration and quality of RNA were measured based on the method described by Cheng et al. [23 (link)]. After that, the first-strand complementary DNA (cDNA) was synthesized from total RNA with HiScript II Q RT Select SuperMix for qPCR(+gDNA wiper) (Vazyme Biotech Co., Ltd., Nanjing, China). The qRT-PCR analysis was performed using ChamQ SYBR qPCR Master Mix according to the recommended process of the manufacturer (Vazyme Biotech Co., Ltd., Nanjing, China). Each sample was tested in duplicate. The 2^−ΔΔCT method was used to calculate the relative mRNA level of target genes after normalization against the reference gene β-actin, while the mRNA expression of each target gene of mice in the CON group was assigned as a value of 1. The primers used for qRT-PCR are presented in Table 1.

HT-29 and SW620 cells were, respectively, treated with OMT (5 mM), DOX (0.3 μM), or OMT + DOX for 48 h. RNA was extracted by using RNA Isolation reagent (Vazyme Biotech, R701) via the standard procedure. Retrotranscription was performed according to the instructions of the EasyScript^® One-Step gDNA Removal and cDNA Synthesis Supermix kit at 42°C for 15 min and then 85°C for 5 s. The quantitative real-time PCR (qPCR) reaction was carried out as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 10 s, and annealing at 60°C for 30 s, for a total of 40 cycles. The primers used for qPCR for SPTAN1 were 5′ GCC​AAC​TCA​GGA​GCC​ATT​GTT-3′ (forward) and 5′- CGG​GTC​CGT​ATG​GTT​TCA​GAT-3′ (reverse); for FHL-2 were 5′ TAC​AGA​CTG​CTA​TTC​CAA​CGA​G-3′ (forward) and 5′- GCA​CTG​CAT​GGC​ATG​TTG​TT-3′ (reverse); for PPA1 were 5′ CAT​ACT​GGC​TGT​TGT​GGT​GAC-3′ (forward) and 5′- GCC​TAG​AAC​TTT​CAC​GCC​AAT-3′ (reverse); for COX 15 were 5′ TCA​CAC​CGA​ATG​TGG​GGT​C-3′ (forward) and 5′- AGA​ACA​CGT​CCT​TTC​ATG​CCA-3′ (reverse); for ACTB were 5′ CAT​GTA​CGT​TGC​TAT​CCA​GGC-3′ (forward) and 5′- CTC​CTT​AAT​GTC​ACG​CAC​GAT-3′ (reverse); and for GAPDH were 5′ CTG​GGC​TAC​ACT​GAG​CAC​C-3′ (forward) and 5′- AAG​TGG​TCG​TTG​AGG​GCA​ATG -3′ (reverse).

Total RNA from colonic biopsies was isolated using RNA isolation reagent (Vazyme, R401-01), and the concentration was measured. Total RNA (1 μg) was reverse transcribed into cDNA using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme, R323-01). Subsequently, cDNA was amplified using the StepOnePlus™ Real-Time PCR System (Thermo Fisher; 4376600). The PCR mixture containing 10 μl of 2× AceQ^®Universal SYBR qPCR Master Mix (Vazyme, Q511-02), 1 μl of cDNA, forward and reverse primers (0.8 μl each), and diethyl pyrocarbonate (DEPC) water (7.4 μl) was prepared. The primers used for qPCR are listed in Table 2. GAPDH was used as the internal standard to normalize target gene expression. For analysis, the 2^−ΔΔCt method was used to calculate the mRNA levels of the target genes.

The total RNAs of liver tissues were easily extracted using an RNA isolation reagent (Nanjing Vazyme, Catalog No. R701) following the manufacturer’s protocol. The cDNA was reverse transcribed from the RNA using HiScript III All-in-one RT SuperMix (Nanjing Vazyme, Catalog No. R333). The real-time reverse transcription–quantitative polymerase chain reaction (RT-qPCR) was performed using ChamQ SYBR Color qPCR Master Mix (Nanjing Vazyme, Catalog No. Q421-02) with 40 cycles of 95 °C for 10 s and 60 °C for 30 s, followed by 95 °C for 15 s and 60 °C for 60 s using RT-qPCR System machine (LightCycler480; Roche, Switzerland). The relative expression levels of target genes were calculated by employing 2^−ΔΔCT method. The sequences of primers used for amplifying the target genes are listed in Table 1.