Abstract
The intestinal mucosal development of piglets (Sus scrofa) during the weaning stage is important to their disease susceptibility and later growth. Quantitative real-time PCR (RT-qPCR) is commonly used to screen for differentially expressed genes and, for accurate results, proper reference housekeeping genes are essential. Here we assessed the mRNA expression of 18 well-known candidate reference genes at different parts of the gastrointestinal tract (GIT) of piglets during the weaning process by RT-qPCR assay. GeNorm analysis revealed that B2M/HMBS/HPRT1 were the three most stable reference genes and GAPDH was the least stable gene in the duodenum, jejunum, ileum, colon, and whole GIT. BestKeeper analysis found that B2M/HMBS/PGK11, HMBS/B2M/HPRT1, B2M/HMBS/HSPCB, B2M/HPRT1/HMBS, and B2M/HMBS/HPRT1 were the most stable genes in the duodenum, jejunum, ileum, colon, and whole GIT, respectively, whereas GAPDH, B-actin, and 18S rRNA were the least stable genes at different parts of the GIT. To confirm the crucial role of appropriate housekeeping genes in obtaining reliable results, we analyzed the expression of ALP using each of the 18 reference genes to normalize the RT-qPCR data. We found that the expression levels of ALP normalized using the most stable reference genes (B2M/HMBS/HPRT1) differed greatly from the expression levels obtained when the data were normalized using the least stable genes (GAPDH, B-actin, and 18S). We concluded that B2M/HMBS/HPRT1 were the optimal reference genes for gene expression analysis by RT-qPCR in the intestinal mucosal development stages of piglets at weaning. Our findings provide a set of porcine housekeeping reference genes for studies of mRNA expression in different parts of the pig intestine.
Highlights
The mRNA expression of all the candidate reference genes was assayed in all the mucosa samples from different parts of the gastrointestinal tract (GIT)
We identified B2M, hydroxymethylbilane synthase (HMBS), and hypoxanthine phosphoribosyltransferase 1 (HPRT1) as the optimal housekeeping genes for normalization of RT-qPCR expression data of genes involved in the intestinal development of piglets because of their high expression abundance and high stability in different parts of the GIT
The results of the GeNorm and BestKeeper analyses indicated that B2M/HMBS/HPRT1 were the optimal reference genes in duodenum, jejunum, ileum, and colon, as well as the whole GIT of pig, whereas other commonly used reference genes (e.g., glyceraldehyde-3-phosphate dehydrogenase (GAPDH), B-actin, and 18S) were the least stable
Summary
Reference genes for normalizing RT-qPCR data from pig intestine. Especially by Quantitative real-time PCR (RT-qPCR), has already provided insights into the potential mechanisms involved in the intestinal mucosal development in pig [6,7]. Unlike other methods used in gene expression studies, RT-qPCR data are generally normalized against a set of housekeeping reference genes to minimize the differences among starting materials [8]. The identification of an appropriate set of housekeeping genes as endogenous reference genes is crucial to normalize RT-qPCR data [10,11]. Ideal housekeeping genes are expected to be expressed at a constant level in different tissues at all developmental stages, be unaffected by experimental treatments, and should pass through the same steps involved in gene analysis for qualification [9]. Information about constant housekeeping genes in the intestinal mucosa of piglets in early life is largely lacking
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