Quantitative PCR Analysis of Digestive Genes

Quantitative PCR was performed in triplicates using a SYBR Green kit SensiFAST™ SYBR® No-ROX (Bioline, Sydney, Australia) with Rotorgene 6000 real-time PCR machine (Corbett Research, Sydney, Australia). The PCR reaction was performed in a volume of 10 μL containing 5 μL of 2× SensiFAST, 400 mM of each primer and 2 μL of cDNA template. After thermal cycling, amplification cycle (Cq) values for all genes were collected and imported into qBase+ version 3.0 (Biogazelle, Zwijnbeke, Belgium) software and analyzed against two optimized reference genes, HPRT1 and TBP, in the current study. Both reference genes were applied to the samples of each tissue. The qBase+ applied an arithmetic mean method to transform logarithmic Cq value to linear relative quantity using exponential function for relative quantification of genes [22 , 23 ] and the output data was exported to SPSS statistics version 22 (IBM SPSS, UK) for further analysis. The relative expression levels of the genes in respective treatment groups are expressed as means of normalized relative quantities (NRQ). Relative quantity for individual genes was scaled to the lowest average expression level of the treatment being 1.
The genes analyzed in the tissues are listed as follow: PGA5, PGC and CCK, in proventriculus; AMY2A, ATP1A1, CCK1R, CCK, CELA1, CELA2A, PNLIP, in pancreas; and APN, ASCT1, ATP1A1, B^oAT, CAT1, CAT2, CCK1R, CCK, EAAT3, b^o,+AT, GLUT1, GLUT2, LAT1, PepT1, PepT2, SI, y⁺LAT1, y⁺LAT2, and rBAT in the intestine.