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. 2018 Sep 20;13(9):e0204348.
doi: 10.1371/journal.pone.0204348. eCollection 2018.

Identification of endogenous normalizing genes for expression studies in inguinal ring tissue for scrotal hernias in pigs

William Raphael Lorenzetti  1   2 , Adriana Mercia Guaratini Ibelli  2   3 , Jane de Oliveira Peixoto  2 , Marcos Antonio Zanella Mores  2 , Igor Ricardo Savoldi  3 , Kamilla Bleil do Carmo  3 , Haniel Cedraz de Oliveira  4 , Mônica Corrêa Ledur  1   2
Affiliations

Identification of endogenous normalizing genes for expression studies in inguinal ring tissue for scrotal hernias in pigs

William Raphael Lorenzetti et al. PLoS One. .

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Cycle threshold (Ct) variation in normal and hernia-affected pigs in the two experiments.
CG: control group; AG: affected group; 1 –experiment 1 and 2 –experiment 2. *PPIA: just the information about the experiment 2 was plotted, since there was no amplification for some samples in the experiment 1.
Fig 2
Fig 2. Melting curve analyzes of the 10 reference candidate genes evaluated in this study.
Fig 3
Fig 3. Ranking of reference candidate genes based on the average expression stability using the geNorm software.
A: results obtained in the Experiment 1. B: results obtained in the Experiment 2.
Fig 4
Fig 4. Suitable genes ranked by the BruteAgreeg tool in the two simulations for Experiment 1.
A: simulation 1, genes RPL19, RPL32 and HMBS; B: simulation 2, genes RPL19, H3F3A and HMBS (Table 3).
Fig 5
Fig 5. Suitable genes ranked by the BruteAgreeg tool in the two simulations for Experiment 2.
A: Simulation 1, genes PPIA, RPL19 and HMBS; B) Simulation 2, genes PPIA, RPL19 and YWHAZ (Table 3).
See this image and copyright information in PMC

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