Intestinal Total RNA Extraction and qPCR Analysis

Total RNA extraction of intestinal tissue and mucosal cells after centrifugation was performed with Trizol (Invitrogen). RNA was quantified using Nanodrop2000 (Thermo Fisher) and its integrity was checked on a 1% agarose gel. cDNA was synthesized from RNA samples (600 ng) using HiScript^® II QRT SuperMix for qPCR with gDNA wiper (Vazyme). Quantitative real-time PCR (qPCR) was performed on a Bio-Rad CFX Maestro™ (Bio-Rad) instrument with 6ng cDNA per well using Hieff^® qPCR SYBR^® Green Master Mix (Yeasen). The run was 95°C for 5 minutes, then 95°C for 10 seconds, 60°C for 15 seconds, 72°C for 60 seconds with 41 cycles. Each reaction was performed in triplicate. The qPCR analysis was performed according to previously published procedures (38 (link), 39 (link)) with rpl13a serving as the endogenous control. The average ΔCt value was calculated by subtracting the control ΔCt value from the treated ΔCt value. The relative amount of mRNA was calculated as 2^-ΔΔCt. The primers used in the current study were partially cited from published articles or designed and tested using the primer BLAST in NCBI. The primers used are summarized in Table 1.