Myiq system software version 1

Intestinal specimens suspended in RNA lysis buffer containing β-mercaptoethanol were homogenized using the TissueLyser (Qiagen, Hilden, Germany) for 1 minute/25 Hz and total RNA was isolated using spin columns based on manufacturer's instructions (Promega, Madison, WI, USA). RNA was reverse-transcribed to cDNA using iScriptTM cDNA Synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). The PCR reaction mixture, containing iQSYBR Green Supermix (Bio-Rad Laboratories Inc.) was prepared based on manufacturer’s instructions and qRT-PCR analysis was performed using the MyiQ single-colour real time PCR detection system (Bio-Rad Laboratories Inc.) with MyiQ System Software Version 1.0.410 (Bio-Rad Laboratories Inc.). Commercially manufactured gene specific primers (Eurogentec, Seraing, Belgium) were used after confirmation of specificity and efficiency tests by qRT-PCR with dilution series of pooled cDNA at a temperature gradient (55°C to 65°C) for primer-annealing and subsequent melting curve analysis (S2 Table). The mRNA quantity was calculated relative to the expression of β-Actin (ACTB) reference gene.

A liver sample was collected from the same sampled birds for jejunum collection (10 chickens per treatment) and submitted to RNA isolation using the SV Total RNA Isolation System (Promega, Madison, WI, USA) and subsequent cDNA Synthesis (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Primers, as presented in Table 9, were commercially produced (Eurogentec, Maastricht, The Netherlands). qPCR was performed using the MyIQ single-color, real-time PCR detection system (Bio-Rad) and MyiQ System Software Version 1.0.410 (Bio Rad Laboratories Inc., Hercules, CA, USA). Data were analyzed using the efficiency-corrected DeltaDelta-Ct method [54 (link)]. The fold-change values of the genes of interest were normalized using the geometric mean of the fold-change values of hypoxanthine-guanine phosphoribosyl transferase (HPRT) and β-actin (ACTB). The mRNA expression of markers in the liver was selected based on their role, i.e., oxidative stress (GSS and iNOS) and metabolism (CPT1 and HMGCR).