Quantitative PCR
Isolation of RNA, cDNA conversion and quantitative real-time PCR from the various intestinal segments (jejunum, ileum, proximal colon and mid-distal colon) were done as per manufacturer’s instructions and as previously described (Tan et al., 2021). Ribosomal protein S9 (RPS9) was used as housekeeping gene. The list of primers used can be found in Supplementary Table 1. The total RNA was extracted with the RNeasy® Mini Kit (Qiagen GmbH, Hilden, Germany) based on the manufacturer’s instructions. 1μg RNA was reverse transcribed using the QuantiTect® Reverse Transcription Kit (Qiagen GmbH, Hilden, Germany). cDNA was then diluted 1:20 with DNase free water, and 4μL of the dilution was used as a template for PCR. Each reaction contained 5μL qPCRBIO SyGreen Mix Lo-ROX (PCR Biosystems, Dueren, Germany), 4 µl template and an appropriate amount of primers.