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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Genomics and Improvement Laboratory » Research » Publications at this Location » Publication #334539

Research Project: Understanding Genetic and Physiological Factors Affecting Nutrient Use Efficiency of Dairy Cattle

Location: Animal Genomics and Improvement Laboratory

Title: Selection of internal reference genes for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in the rumen epithelium

Author
item Die, Jose - Collaborator
item Baldwin, Ransom - Randy
item Rowland, Lisa
item Li, Robert
item Oh, Sunghee - Jeju National University
item Li, Congjun
item Connor, Erin
item Ranilla, Maria-jose - University Of Leon

Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/8/2017
Publication Date: 2/24/2017
Citation: Die, J.V., Baldwin, R.L., Rowland, L.J., Li, R.W., Oh, S., Li, C., Connor, E.E., Ranilla, M. 2017. Selection of internal reference genes for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in the rumen epithelium. PLoS One. 12(2):e0172674.

Interpretive Summary: Rumen epithelium is an important tissue of the digestive tract of cows. In order to study this tissue and evaluate the changes that occur in response to dietary changes, we need to have markers that are consistent to allow for better comparisons between animals. Reference genes are used in the study of gene expression experiments to serve as markers. This paper discusses the selection process we used to define 8 markers for rumen epithelium genes for references and discusses why five of these markers will be very useful in future studies.

Technical Abstract: The rumen is lined on the luminal side by a stratified squamous epithelium that is responsible for not only absorption, but also transport, extensive short-chain fatty acid (SCFA) metabolism and protection. Butyrate has been demonstrated to initiate the differentiation of the tissue following introduction of solid feed to the weaning neonate as well as affecting the metabolism of other nutrients and absorption of nutrients in in vitro experiments. The objective of the present study was to validate expression stability of eight putative reference genes bovine rumen, considering the intrinsic heterogeneity of bovine rumen with regard to different luminal characteristics due to direct infusion of butyrate to double the intra-ruminal content of the rumen liquor. Our focus was on identifying stable reference genes which are suitable to normalize real-time RT-qPCR experiments from rumen samples collected from clinical assays, irrespective of localization within the organ and the across physiological state. The most stably expressed genes included: ACTBF, UXTF, DBNDD2F, RPS9F, DDX54F and HMBSF. Their high stability values suggest these reference genes will facilitate better evaluation of variation of across an array of conditions including: localization within the rumen, differences among cattle fed an array of rations, as well as response to development in the weaning animal. Moreover, we anticipate these reference genes may be useful for expression studies in other ruminants.