title: Selection of reference genes for quantitative real-time polymerase chain reaction in porcine embryos.
abstract: To study gene expression and to determine distinctive characteristics of embryos produced by different methods, normalisation of the gene(s) of interest against reference gene(s) has commonly been employed. Therefore, the present study aimed to assess which reference genes tend to express more stably in single porcine blastocysts produced in vivo (IVO) or by parthenogenetic activation (PA), in vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT) using different analysis programs, namely geNorm, Normfinder and Bestkeeper. Commonly used reference genes including 18S rRNA (18S), H2A histone family, member Z (H2A), hypoxanthine phosphoribosyltransferase1 (HPRT1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein 4 (RPL4), peptidylprolyl isomerase A (PPIA), beta actin (ACTB), succinate dehydrogenase complex, subunit A (SDHA) and hydroxymethylbilane synthase (HMBS2) were analysed; most of them resulted in significantly (P<0.05) different cycle threshold (CT) values in porcine embryos except for SDHA and H2A. In evaluation of stable reference genes across in vivo and in vitro porcine blastocysts, three kinds of programs showed slightly different results; however, there were similar patterns about the rankings of more or less stability overall. In conclusion, SDHA and H2A were determined as the most appropriate reference genes for reliable normalisation in order to find the comparative gene expression in porcine blastocysts produced by different methods, whereas 18S was regarded as a less-stable reference gene. The present study has evaluated the stability of commonly used reference genes for accurate normalisation in porcine embryos to obtain reliable results.
authors: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, 501, Jinju-daero, Jinju 660-701, Republic of Korea.
authors: Won-Jae
authors: WJ
authors: Lee
authors: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, 501, Jinju-daero, Jinju 660-701, Republic of Korea.
authors: Si-Jung
authors: SJ
authors: Jang
authors: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, 501, Jinju-daero, Jinju 660-701, Republic of Korea.
authors: Seung-Chan
authors: SC
authors: Lee
authors: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, 501, Jinju-daero, Jinju 660-701, Republic of Korea.
authors: Ji-Sung
authors: JS
authors: Park
authors: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, 501, Jinju-daero, Jinju 660-701, Republic of Korea.
authors: Ryoung-Hoon
authors: RH
authors: Jeon
authors: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, 501, Jinju-daero, Jinju 660-701, Republic of Korea.
authors: Raghavendra Baregundi
authors: RB
authors: Subbarao
authors: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, 501, Jinju-daero, Jinju 660-701, Republic of Korea.
authors: Dinesh
authors: D
authors: Bharti
authors: Department of Obstetrics and Gynecology, School of Medicine and Institute of Health Science, Gyeongsang National University, Jinju 660-701, Republic of Korea.
authors: Jeong-Kyu
authors: JK
authors: Shin
authors: Department of Oral and Maxillofacial Surgery, Institute of Health Science, School of Medicine, Gyeongsang National University, Jinju 660-701, Republic of Korea.
authors: Bong-Wook
authors: BW
authors: Park
authors: Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, 501, Jinju-daero, Jinju 660-701, Republic of Korea.
authors: Gyu-Jin
authors: GJ
authors: Rho
mesh_terms: D000818:Animals; D004622:Embryo, Mammalian / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D020043:Genes, Essential; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013552:Swine