Computational protocol: The Expression of Type-1 and Type-2 Nitric Oxide Synthase in Selected Tissues of the Gastrointestinal Tract during Mixed Mycotoxicosis

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Protocol publication

[…] Real-Time PCR was performed in the Genomics and Transcriptomics Laboratory of the Department of Animal Anatomy, Faculty of Veterinary Medicine of the University of Warmia and Mazury in Olsztyn. Real-Time PCR was carried out to determine the mRNA expression levels of NOS-2 and NOS-1 genes in reference to the GAPDH gene. Specific primers for the above genes were designed in the Pick Primer Blast application based on the following sequences: NOS-2–NM_001143690.1, NOS-1–XM_003132898.3 and GAPDH–NM_001206359.1 (). Reaction tubes were filled with 25 μL of the reaction mix, and the assay was performed in the 7500 Fast Real-Time PCR System thermocycler (Applied Biosystems, Carlsbad, California USA) under the following conditions: initial denaturation −10 min/95 °C, followed by 40 cycles: denaturation −15 s/95 °C, primer annealing −1 min/60 °C. The following reagents were used: 12.5 μL of FastStart Universal SYBR Green Master (Rox) (Roche, Vaud, Switzerland), 10.5 μL of nuclease-free water, 1 μL of cDNA, 1 μL of the primer mix, 5 µM each (forward + reverse). All samples were analyzed in duplicates. [...] The data were grouped based on: (i) the administered mycotoxins relative to experimental dates and (ii) experimental dates relative to the analyzed mycotoxins. Differences between the administered mycotoxins relative to experimental dates and differences between experimental dates relative to the analyzed mycotoxins were analyzed. The results were processed statistically in the Statistica application. Differences between groups (mycotoxin or date) were determined by ANOVA. The equality of group variances was tested by the Brown-Forsythe test. When ANOVA revealed significant differences between groups (P < 0.01—highly significant differences, 0.01 < P < 0.05—significant differences, P > 0.05—no differences), Tukey’s HSD test was used to determine differences between specific groups. […]

Pipeline specifications

Software tools Primer-BLAST, Statistica
Applications Miscellaneous, qPCR
Diseases Mycotoxicosis
Chemicals Nitric Oxide, Zearalenone