A New Set of Reference Housekeeping Genes for the Normalization RT-qPCR Data from the Intestine of Piglets During Weaning

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2018 Sep 27

PMID 30256841

Citations 23

Authors

Shujin Wang

Binxing Wang

Huan He

Aomin Sun

Chunhua Guo

Affiliations

Soon will be listed here.

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.

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References

Facci M, Auray G, Meurens F, Buchanan R, van Kessel J, Gerdts V . Stability of expression of reference genes in porcine peripheral blood mononuclear and dendritic cells. Vet Immunol Immunopathol. 2011; 141(1-2):11-5. DOI: 10.1016/j.vetimm.2011.01.005. View

Erkens T, Van Poucke M, Vandesompele J, Goossens K, Van Zeveren A, Peelman L . Development of a new set of reference genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC1A. BMC Biotechnol. 2006; 6:41. PMC: 1609116. DOI: 10.1186/1472-6750-6-41. View

Wang Y, Zhao Y, Li J, Liu H, Ernst C, Liu X . Evaluation of housekeeping genes for normalizing real-time quantitative PCR assays in pig skeletal muscle at multiple developmental stages. Gene. 2015; 565(2):235-41. DOI: 10.1016/j.gene.2015.04.016. View

Gu Y, Li M, Zhang K, Chen L, Jiang A, Wang J . Evaluation of endogenous control genes for gene expression studies across multiple tissues and in the specific sets of fat- and muscle-type samples of the pig. J Anim Breed Genet. 2011; 128(4):319-25. DOI: 10.1111/j.1439-0388.2011.00920.x. View

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3(7):RESEARCH0034. PMC: 126239. DOI: 10.1186/gb-2002-3-7-research0034. View

Wong M, Medrano J . Real-time PCR for mRNA quantitation. Biotechniques. 2005; 39(1):75-85. DOI: 10.2144/05391RV01. View

Barbara G . Mucosal barrier defects in irritable bowel syndrome. Who left the door open?. Am J Gastroenterol. 2006; 101(6):1295-8. DOI: 10.1111/j.1572-0241.2006.00667.x. View

Park S, Kwon S, Hwang J, Park D, Kim T, Kim C . Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs. Gene. 2015; 558(1):152-8. DOI: 10.1016/j.gene.2014.12.052. View

Bruge F, Venditti E, Tiano L, Littarru G, Damiani E . Reference gene validation for qPCR on normoxia- and hypoxia-cultured human dermal fibroblasts exposed to UVA: is β-actin a reliable normalizer for photoaging studies?. J Biotechnol. 2011; 156(3):153-62. DOI: 10.1016/j.jbiotec.2011.09.018. View

10.

Cinar M, Islam M, Uddin M, Tholen E, Tesfaye D, Looft C . Evaluation of suitable reference genes for gene expression studies in porcine alveolar macrophages in response to LPS and LTA. BMC Res Notes. 2012; 5:107. PMC: 3306271. DOI: 10.1186/1756-0500-5-107. View

11.

Martino A, Cabiati M, Campan M, Prescimone T, Minocci D, Caselli C . Selection of reference genes for normalization of real-time PCR data in minipig heart failure model and evaluation of TNF-α mRNA expression. J Biotechnol. 2011; 153(3-4):92-9. DOI: 10.1016/j.jbiotec.2011.04.002. View

12.

Zhang J, Tang Z, Wang N, Long L, Li K . Evaluating a set of reference genes for expression normalization in multiple tissues and skeletal muscle at different development stages in pigs using quantitative real-time polymerase chain reaction. DNA Cell Biol. 2011; 31(1):106-13. DOI: 10.1089/dna.2011.1249. View

13.

Schmittgen T, Livak K . Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008; 3(6):1101-8. DOI: 10.1038/nprot.2008.73. View

14.

Guibinga G, Hsu S, Friedmann T . Deficiency of the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) dysregulates neurogenesis. Mol Ther. 2009; 18(1):54-62. PMC: 2839227. DOI: 10.1038/mt.2009.178. View

15.

Boudry G, Peron V, Le Huerou-Luron I, Lalles J, Seve B . Weaning induces both transient and long-lasting modifications of absorptive, secretory, and barrier properties of piglet intestine. J Nutr. 2004; 134(9):2256-62. DOI: 10.1093/jn/134.9.2256. View

16.

Svobodova K, Bilek K, Knoll A . Verification of reference genes for relative quantification of gene expression by real-time reverse transcription PCR in the pig. J Appl Genet. 2008; 49(3):263-5. DOI: 10.1007/BF03195623. View

17.

Wang S, Guo C, Zhou L, Zhang Z, Huang Y, Yang J . Comparison of the biological activities of Saccharomyces cerevisiae-expressed intracellular EGF, extracellular EGF, and tagged EGF in early-weaned pigs. Appl Microbiol Biotechnol. 2015; 99(17):7125-35. DOI: 10.1007/s00253-015-6468-6. View

18.

Murani E, Muraniova M, Ponsuksili S, Schellander K, Wimmers K . Identification of genes differentially expressed during prenatal development of skeletal muscle in two pig breeds differing in muscularity. BMC Dev Biol. 2007; 7:109. PMC: 2147030. DOI: 10.1186/1471-213X-7-109. View

19.

Li Q, Domig K, Ettle T, Windisch W, Mair C, Schedle K . Evaluation of potential reference genes for relative quantification by RT-qPCR in different porcine tissues derived from feeding studies. Int J Mol Sci. 2011; 12(3):1727-34. PMC: 3111629. DOI: 10.3390/ijms12031727. View

20.

Kuijk E, du Puy L, van Tol H, Haagsman H, Colenbrander B, Roelen B . Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos. BMC Dev Biol. 2007; 7:58. PMC: 1896162. DOI: 10.1186/1471-213X-7-58. View