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

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.

Transcriptome analysis identifies genes involved with the development of umbilical hernias in pigs

“…For validation, the following 12 DE genes were chosen according to their functions: Matrix metallopeptidase 13 (MMP13), Vitrin (VIT), Alkaline ceramidase 2 (ACER2), Molecule CD3D (CD3D), Galactin 3 (LGALS3), Fos proto-oncogene, AP-1 transcription factor subunit (FOS), collagen and calcium binding EGF domains 1 (CCBE1), Plakophilin 3 (PKP3), Epiphycan (EPYC), S100 calcium binding protein A2 (S100A2), Aggrecan (ACAN) and Microtubule associated protein 1 light chain 3 gamma (MAP1LC3C). In addition, ten candidate reference genes were tested to select the appropriate reference genes to be used in the qPCR analysis (Table 1), as described by [22]. The primers were designed in exon-exon regions using primer-blast tool [23] and their quality was evaluated in the NetPrimer program (http://www.premierbiosoft.…”

Identification of Appropriate Housekeeping Genes for Gene Expression Studies in Human Renal Cell Carcinoma Under Hypoxic Conditions

“…Accurate relative quanti cation in gene expression analysis requires the use of normalized reference genes, since the stability of target genes could vary according to the experimental design, making it essential for the reliability of the results [10,13,14]. The selection of suitable reference genes for gene expression analyses has recently been highlighted in several studies [17][18][19][20][21].…”

Determination of novel reference genes for improving gene expression data normalization in selected canine reproductive tissues – a multistudy analysis

“…It needs to be mentioned that this molecule, a component of the large ribosomal 60S unit, has been considered before as a reference gene for data normalization. Its high stability has been discussed for application to various tissues in, e.g., pig [30,31], rat [32], human [33] and chicken [34,35], and even in several invertebrates [36]. Notably, it was found to be stably expressed in several canine healthy and diseased, i.e., neoplastic, tissues [9,37,38], and was therefore proposed as a good reference gene.…”