Computational protocol: Vitamin D Receptor Expression in Dogs

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Protocol publication

[…] Real‐time qPCR experiments were performed in a Roche Light Cycler 480 with a 12 µL reaction volume containing 4.5 µL cDNA, 1.25 µL primers, and 6.25 µL SYBR Green qPCR Master Mix. Reactions were also performed without reverse transcriptase and without template, using distilled deionized H2O to maintain volume to monitor for contamination. A standard cycling program was used. Samples were run at 50°C for 2 minutes, 95°C for 2 minutes, and then 40 cycles of 95°C for 15 seconds followed by 60°C for 30 seconds.Primers for the VDR, 25‐hydroxyvitamin D3 1‐alpha‐hydroxylase (CYP27B1), ECAD, and CLD2 (Table ) were designed by the Roche primer design software based on canine sequences from the Ensembl database as previously described, so that the predicted amplicon would span exon‐exon boundaries. The primers were assessed by Basic Local Alignment Search Tool analysis (National Center for Biotechnology Information). Sequences were tested with nucleic acid folding software (OligoAnalyser 3.1) in concordance with MIQE guidelines. Primer sequences used for glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), actin beta (ACTB), and Succinate dehydrogenase complex subunit A (SDHA) were previously described (Table )., Specificity of the products was verified for each target and referenced with gel electrophoresis showing a single product of the desired length. In addition, a melting curve analysis was performed for each reference and target.Multiple reference genes were selected in line with available guidelines. Reference genes (Table ) were selected based on previous evidence of high expression within the canine duodenum. Analysis of the reference genes' relative expression levels by the relative expression software tool (REST) (M. Pfaffl [Technical University Munich] and QIAGEN http://www.gene-quantification.de/rest-2009.html) identified no significant differences among groups (control, CE, and PLE). Reference gene expression was also shown to be stable using Best Keeper software. To minimize any technical, run‐to‐run variation between different samples for the comparison of gene expression, the maximum number of samples (in triplicate) were analyzed in each run. As not all samples could be analyzed for 1 gene in the same run, control, and affected cases were spread across 2 runs equally and inter‐run calibrators were included. A correction factor was then generated to control for inter‐run differences. […]

Pipeline specifications

Software tools BLASTN, REST
Application qPCR
Organisms Canis lupus familiaris, Homo sapiens
Diseases Intestinal Diseases
Chemicals Vitamin D