title: Identification of reference genes for relative quantification of circulating microRNAs in bovine serum.
abstract: Circulating microRNAs in body fluids have been implicated as promising biomarkers for physiopathology disorders. Currently, the expression levels of circulating microRNAs are estimated by reverse transcription quantitative real-time polymerase chain reaction. Use of appropriate reference microRNAs for normalization is critical for accurate microRNA expression analysis. However, no study has systematically investigated reference genes for evaluating circulating microRNA expression in cattle. In this study, we describe the identification and characterization of appropriate reference microRNAs for use in the normalization of circulating microRNA levels in bovine serum. We evaluated the expression stability of ten candidate reference genes in bovine serum by using reverse transcription quantitative real-time polymerase chain reaction. Data were analyzed using geNorm, NormFinder, and BestKeeper statistical algorithms. The results consistently showed that a combination of miR-93 and miR-127 provided the most stably expressed reference. The suitability of these microRNAs was validated, and even when compared among different genders or breeds, the combination of miR-93 and miR-127 was ranked as the most stable microRNA reference. Therefore, we conclude that this combination is the optimal endogenous reference for reverse transcription quantitative real-time polymerase chain reaction-based detection of microRNAs in bovine serum. The data presented in this study are crucial to successful biomarker discovery and validation for the diagnosis of physiopathological conditions in cattle.
authors.affiliation: Department of Biology, Kyung Hee University, Seoul, Republic of Korea.
authors.forename: In-Seon
authors.initials: IS
authors.lastname: Bae
authors.affiliation: National Institute of Animal Science, RDA, Pyeongchang, Gangwon, Republic of Korea.
authors.forename: Ki Yong
authors.initials: KY
authors.lastname: Chung
authors.affiliation: Department of Biology, Kyung Hee University, Seoul, Republic of Korea.
authors.forename: Jongmin
authors.initials: J
authors.lastname: Yi
authors.affiliation: National Institute of Animal Science, RDA, Pyeongchang, Gangwon, Republic of Korea.
authors.forename: Tae Il
authors.initials: TI
authors.lastname: Kim
authors.affiliation: Department of Biomedical Laboratory Science, Shinhan University, Uijeongbu, Gyeonggi, Republic of Korea.
authors.forename: Hwa-Sik
authors.initials: HS
authors.lastname: Choi
authors.affiliation: National Institute of Animal Science, RDA, Pyeongchang, Gangwon, Republic of Korea.
authors.forename: Young-Moo
authors.initials: YM
authors.lastname: Cho
authors.affiliation: Bovine Genome Resources Bank, School of Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk, Republic of Korea.
authors.forename: Inho
authors.initials: I
authors.lastname: Choi
authors.affiliation: Department of Biology, Kyung Hee University, Seoul, Republic of Korea.
authors.forename: Sang Hoon
authors.initials: SH
authors.lastname: Kim
mesh_terms: D000818:Animals; D002417:Cattle; D006461:Hemolysis; D035683:MicroRNAs / Q000097:blood* / Q000235:genetics
chemical_list: D035683:MicroRNAs
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