Therlands) (Table 1). The primers used were selected based on specificity and

Therlands) (Table 1). The primers used were selected based on specificity and efficiency by qPCR analysis of a dilution series of pooled cDNA at a temperature gradient (55uC to 65uC) for primer-annealing and subsequent melting curve analysis. The reaction mixture for the qPCR containing 10 mL of the diluted cDNA was mixed with 15 mL iQSYBR Green Title Loaded From File Supermix (Biorad), forward and Title Loaded From File Reverse primers (final concentration of 0.4 pmol/mL for each primer) and sterile water according to the manufacturer’s instructions. qPCR was performed using the MyiQ single-colour real-time PCR detection system (Biorad) and MyiQ System Software Version 1.0.410 (Biorad). Amplification efficiency was determined per plate using linregPCR. Data were analyzed using the efficiency corrected Delta-Delta-Ct method [27]. Housekeeping genes were tested for all the test conditions after which most stable housekeeping genes for liver and intestinal samples were selected using the geNorm software (data not shown). The most stable housekeeping genes had a M-value between 0.2 and 0.5. To determine if the inclusion of an additional housekeeping gene was required, the cut-off value for variation was set at 0.2. The fold-change values of the genes of interest (GOIs) were normalized using two housekeeping genes: hypoxanthine-guanine phosphoribosyl transferase (HPRT) and hexose-6phosphate dehydrogenase (H6PD). The mRNA expression of proteins involved in oxidative stress i.e. HMOX, HIF-1a and XOR were evaluated in the liver and intestine. Furthermore, mRNA expression of the tight junctions proteins claudin 1 and 5 (CLDN1 and CLDN5) and zona occludens 1 and 2 (ZO1 and ZO2) in sections from the duodenum, jejunum and ileum were measured. Two compounds of the immune system, namely TollMaterials and Methods Ethics StatementThe protocol was approved by the Ethical Committee of the Faculty of Veterinary Medicine (Ghent University) (EC 2010/064 and EC 2010/076). All husbandry practices and euthanasia were performed with full consideration of animal welfare.Animals and DietsThe animals and the experimental design have been described elsewhere [12]. In brief, 32 1-day-old broiler chickens were fedAdsorbing Agent Shifts the Effects of DONTable 1. Primers used for the quantification of housekeeping genes (HKG) and genes of interest (GOI).Gene HKG HPRTAccession NPrimerSequenceProduct size (pb)Annealing TNM_204848.Forward Reverse59 CGTTGCTGTCTCTACTTAAGCAG 39 59 GATATCCCACACTTCGAGGAG 39 59 GGAGAACCAGCACTTCTTAGAC 39 59 GGGTTCAGCAATTCCACTGH6PDXM_425746.Forward ReverseGOI CLDN1 NM_001013611 Forward Reverse CLDN5 NM_204201 Forward Reverse HIF-1a NM_204297 Forward Reverse HMOX NM_205344 Forward Reverse TLR2 NM_204278 Forward Reverse TLR4 NM_001030693 Forward Reverse XOR NM_205127 Forward Reverse ZO1 XM_413773 Forward Reverse ZO2 NM_204918 Forward Reverse doi:10.1371/journal.pone.0069014.t001 59 CTGATTGCTTCCAACCAG 39 59 CAGGTCAAACAGAGGTACAAG 39 59 CATCACTTCTCCTTCGTCAGC 39 59 GCACAAAGATCTCCCAGGTC 39 59 CACCATTACCATACTTCAGCAG 39 59 CTTCACATCATCCACACGTTC 39 59 CTTGGCACAAGGAGTGTTAAC 39 59 CATCCTGCTTGTCCTCTCAC 39 59 CCTGCAACGGTCATCTCAG 39 59 GTCTCAGGGCTTGTTCTTCAG 39 59 CTGACCTACCCATCGGACAC 39 59 GCCTGAGAGAGGTCAGGTTG 39 59 GTGTCGGTGTACAGGATACAGAC 39 59 CCTTACTATGACAGCATCCAGTG 39 59 CTTCAGGTGTTTCTCTTCCTCCTC 39 59 CTGTGGTTTCATGGCTGGATC 39 59 CGGCAGCTATCAGACCACTC 39 59 CACAGACCAGCAAGCCTACAG 39 87 64?5 131 59 110 61 111 59 135 59 78 61?3 88 65 111 56?5 140 57?like receptors (TLR) 2 and 4 were also investigated duri.Therlands) (Table 1). The primers used were selected based on specificity and efficiency by qPCR analysis of a dilution series of pooled cDNA at a temperature gradient (55uC to 65uC) for primer-annealing and subsequent melting curve analysis. The reaction mixture for the qPCR containing 10 mL of the diluted cDNA was mixed with 15 mL iQSYBR Green Supermix (Biorad), forward and reverse primers (final concentration of 0.4 pmol/mL for each primer) and sterile water according to the manufacturer’s instructions. qPCR was performed using the MyiQ single-colour real-time PCR detection system (Biorad) and MyiQ System Software Version 1.0.410 (Biorad). Amplification efficiency was determined per plate using linregPCR. Data were analyzed using the efficiency corrected Delta-Delta-Ct method [27]. Housekeeping genes were tested for all the test conditions after which most stable housekeeping genes for liver and intestinal samples were selected using the geNorm software (data not shown). The most stable housekeeping genes had a M-value between 0.2 and 0.5. To determine if the inclusion of an additional housekeeping gene was required, the cut-off value for variation was set at 0.2. The fold-change values of the genes of interest (GOIs) were normalized using two housekeeping genes: hypoxanthine-guanine phosphoribosyl transferase (HPRT) and hexose-6phosphate dehydrogenase (H6PD). The mRNA expression of proteins involved in oxidative stress i.e. HMOX, HIF-1a and XOR were evaluated in the liver and intestine. Furthermore, mRNA expression of the tight junctions proteins claudin 1 and 5 (CLDN1 and CLDN5) and zona occludens 1 and 2 (ZO1 and ZO2) in sections from the duodenum, jejunum and ileum were measured. Two compounds of the immune system, namely TollMaterials and Methods Ethics StatementThe protocol was approved by the Ethical Committee of the Faculty of Veterinary Medicine (Ghent University) (EC 2010/064 and EC 2010/076). All husbandry practices and euthanasia were performed with full consideration of animal welfare.Animals and DietsThe animals and the experimental design have been described elsewhere [12]. In brief, 32 1-day-old broiler chickens were fedAdsorbing Agent Shifts the Effects of DONTable 1. Primers used for the quantification of housekeeping genes (HKG) and genes of interest (GOI).Gene HKG HPRTAccession NPrimerSequenceProduct size (pb)Annealing TNM_204848.Forward Reverse59 CGTTGCTGTCTCTACTTAAGCAG 39 59 GATATCCCACACTTCGAGGAG 39 59 GGAGAACCAGCACTTCTTAGAC 39 59 GGGTTCAGCAATTCCACTGH6PDXM_425746.Forward ReverseGOI CLDN1 NM_001013611 Forward Reverse CLDN5 NM_204201 Forward Reverse HIF-1a NM_204297 Forward Reverse HMOX NM_205344 Forward Reverse TLR2 NM_204278 Forward Reverse TLR4 NM_001030693 Forward Reverse XOR NM_205127 Forward Reverse ZO1 XM_413773 Forward Reverse ZO2 NM_204918 Forward Reverse doi:10.1371/journal.pone.0069014.t001 59 CTGATTGCTTCCAACCAG 39 59 CAGGTCAAACAGAGGTACAAG 39 59 CATCACTTCTCCTTCGTCAGC 39 59 GCACAAAGATCTCCCAGGTC 39 59 CACCATTACCATACTTCAGCAG 39 59 CTTCACATCATCCACACGTTC 39 59 CTTGGCACAAGGAGTGTTAAC 39 59 CATCCTGCTTGTCCTCTCAC 39 59 CCTGCAACGGTCATCTCAG 39 59 GTCTCAGGGCTTGTTCTTCAG 39 59 CTGACCTACCCATCGGACAC 39 59 GCCTGAGAGAGGTCAGGTTG 39 59 GTGTCGGTGTACAGGATACAGAC 39 59 CCTTACTATGACAGCATCCAGTG 39 59 CTTCAGGTGTTTCTCTTCCTCCTC 39 59 CTGTGGTTTCATGGCTGGATC 39 59 CGGCAGCTATCAGACCACTC 39 59 CACAGACCAGCAAGCCTACAG 39 87 64?5 131 59 110 61 111 59 135 59 78 61?3 88 65 111 56?5 140 57?like receptors (TLR) 2 and 4 were also investigated duri.