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.