Abstract
The use of reference genes is required for relative quantification in gene expression analysis and since the stability of these genes could be variable depending on the experimental design, it has become indispensable to test the reliability of endogenous genes. Therefore, this study evaluated 10 reference candidate genes in two different experimental conditions in order to obtain stable genes to be used as reference in expression studies related to scrotal hernias in pigs. Two independent experiments were performed: one with 30 days-old MS115 pigs and the other with 60 days-old Landrace pigs. The inguinal ring/canal was collected, frozen and further submitted to real-time PCR analysis (qPCR). For the reference genes stability evaluation, four tools were used: GeNorm in the SLqPCR, BestKeeper, NormFinder and Comparative CT. A general ranking was generated using the BruteAggreg function of R environment. In this study, the RPL19 was one of the most reliable endogenous genes for both experiments. The breed/age effects influenced the expression stability of candidate reference genes evaluated in the inguinal ring of pigs. Therefore, this study reinforces the importance of evaluating the stability of several endogenous genes previous their use, since a consensual set of reference genes is not easily obtained. Here, two sets of genes are recommended: RPL19, RPL32 and H3F3A for 30-days MS115 and PPIA and RPL19 for the 60 days-old Landrace pigs. This is the first study using the inguinal ring tissue and the results can be useful as an indicative for other studies working with gene expression in this tissue.
Highlights
The real time PCR is one of the main approaches used for gene expression studies, being highly sensitive [1]
Regarding the RNA quality, the average A260/280 ratio was 1.90 ± 0.04 and 2.06 ± 0.01 for the unaffected pig samples and 1.92 ± 0.05 and 2.07 ± 0.02 for herniated pig samples in the Experiment 1 (E1) and Experiment 2 (E2), respectively, evidencing a good quality of the RNA samples to be used in the further analysis
The peptidyl prolyl cis-trans isomerase A (PPIA) gene was removed from the Experiment 1 analysis since there was no amplification for some of the samples, differing from the Experiment 2, where all samples amplified for this gene, with average Ct mean of 15.98 ± 0.34 and the smallest standard deviation
Summary
The real time PCR (qPCR) is one of the main approaches used for gene expression studies, being highly sensitive [1]. Endogenous genes in the inguinal ring tissue for scrotal hernia expression studies in pigs funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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