Duodenal Gene Expression Analysis

To obtain the cDNA, a total of 20 µL reverse transcriptase (RT) reaction was carried out in a BioRad C1000 touch thermocycler using the Improm-II Reverse Transcriptase Kit (Catalog #A1250; Promega, Madison, WI, USA). The concentration of the resulting cDNA was determined by measuring the absorbance at 260 nm and 280 nm, applying an extinction coefficient of 33 (for single-stranded DNA).
For gene expression analysis of the duodenum, a real-time polymerase chain reaction (RT-PCR) was conducted. The primers used in the real-time qPCR were designed based on gene sequences sourced from the Genbank database, utilizing the Real-Time Primer Design Tool software (https://www.idtdna.com/scitools/Applications/RealTimePCR/default.aspx) (IDT DNA, Coralvilla, IA, USA) [29 (link)]. The sequences and descriptions of the primers used can be found in Table 1. To assess primer specificity, a BLAST search against the genomic National Center for Biotechnology Information (NCBI) database was performed. The primer for Gallus gallus 18S rRNA was designed as a reference gene.