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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.

A new set of reference housekeeping genes for the normalization RT-qPCR data from the intestine of piglets during weaning