Gene Expression Analysis in Gut Tissues

The total RNA of liver, duodenum, jejunum, and ileum was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The concentration of RNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and its integrity verified by electrophoresis on a 1% agarose gel. The cDNA was reverse transcribed from 1 μg of total RNA using the Revert Aid Reverse Transcriptase (Takara, Japan), then used for evaluating gene expression. The primers used for target genes are presented in Table 2. The qPCR was performed in a 10-μL reaction volume including 0.5 μM of each forward and reverse primer, 2 μL of cDNA, 2 μL of DEPC treated water, and 5 μL of SYBR Premix Ex Taq (Takara Bio Inc., Japan). The relative expression levels of genes were performed using the Lightcycler-480II system (Roche Diagnostics GmbH, Mannheim, Germany). The PCR cycling condition was 40 cycles at 94 °C for 40 s, 60 °C for 30 s and 72 °C for 35 s. Zn-Met group served as the control group and ZnSO₄, _feed and ZnSO₄, _water groups served as treatment. The relative expression was expressed using the formula 2^{−(∆∆Ct)}, where ∆∆Ct = (Ct_Target − Ct_β-actin)_treatment − (Ct_Target − Ct_β-actin)_control [16 (link),17 (link)]. Relative expression was normalized and expressed as a ratio to the expression in the Zn-Met group.