title: Selection of internal reference genes for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in the rumen epithelium.
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: ACTB, UXT, DBNDD2, RPS9, DDX54 and HMBS. 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.
authors.affiliation: Genetic Improvement of Fruits and Vegetables Laboratory, US Department of Agriculture, Agricultural Research Service, Beltsville, Maryland, United States of America.
authors.forename: Jose V
authors.initials: JV
authors.lastname: Die
authors.affiliation: Animal Genomics and Improvement Laboratory, US Department of Agriculture, Agricultural Research Service, Beltsville, Maryland, United States of America.
authors.forename: Ransom L
authors.identifier: http://orcid.org/0000-0002-0753-4377
authors.initials: RL
authors.lastname: Baldwin
authors.affiliation: Genetic Improvement of Fruits and Vegetables Laboratory, US Department of Agriculture, Agricultural Research Service, Beltsville, Maryland, United States of America.
authors.forename: Lisa J
authors.initials: LJ
authors.lastname: Rowland
authors.affiliation: Animal Genomics and Improvement Laboratory, US Department of Agriculture, Agricultural Research Service, Beltsville, Maryland, United States of America.
authors.forename: Robert
authors.initials: R
authors.lastname: Li
authors.affiliation: Dept. of Computer Science & Statistics, Jeju National University, Jeju City, Jeju Do, S. Korea.
authors.forename: Sunghee
authors.initials: S
authors.lastname: Oh
authors.affiliation: Animal Genomics and Improvement Laboratory, US Department of Agriculture, Agricultural Research Service, Beltsville, Maryland, United States of America.
authors.forename: Congjun
authors.initials: C
authors.lastname: Li
authors.affiliation: Animal Genomics and Improvement Laboratory, US Department of Agriculture, Agricultural Research Service, Beltsville, Maryland, United States of America.
authors.forename: Erin E
authors.initials: EE
authors.lastname: Connor
authors.affiliation: Departamento de Producción Animal, Universidad de León, León, Spain.
authors.forename: Maria-Jose
authors.initials: MJ
authors.lastname: Ranilla
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002087:Butyrates / Q000378:metabolism / Q000494:pharmacology*; D002417:Cattle; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D049029:Dystrophin-Associated Proteins / Q000235:genetics / Q000378:metabolism; D004848:Epithelium / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D005260:Female; D065569:Gastrointestinal Absorption / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D020043:Genes, Essential; D011163:Hydroxymethylbilane Synthase / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D012417:Rumen / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D014886:Weaning
chemical_list: D000199:Actins; D002087:Butyrates; D049029:Dystrophin-Associated Proteins; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins; D011163:Hydroxymethylbilane Synthase; D053487:DEAD-box RNA Helicases