Selection of reference genes for quantitative real-time polymerase chain reaction in porcine embryos
Won-Jae Lee A B , Si-Jung Jang A , Seung-Chan Lee A , Ji-Sung Park A , Ryoung-Hoon Jeon A , Raghavendra Baregundi Subbarao A , Dinesh Bharti A , Jeong-Kyu Shin C , Bong-Wook Park D and Gyu-Jin Rho A E F
+ Author Affiliations
- Author Affiliations
A Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, 501, Jinju-daero, Jinju 660-701, Republic of Korea.
B PWG Genetics Pvt. Ltd, 15 Tech Park Crescent, 638117, Singapore.
C Department of Obstetrics and Gynecology, School of Medicine and Institute of Health Science, Gyeongsang National University, Jinju 660-701, Republic of Korea.
D Department of Oral and Maxillofacial Surgery, Institute of Health Science, School of Medicine, Gyeongsang National University, Jinju 660-701, Republic of Korea.
E Research Institute of Life Sciences, Gyeongsang National University, 501, Jinju-daero, Jinju 660-701, Republic of Korea.
F Corresponding author. Email: jinrho@gnu.ac.kr
*These authors contributed equally to this work.
Reproduction, Fertility and Development 29(2) 357-367 https://doi.org/10.1071/RD14393
Submitted: 15 October 2014 Accepted: 14 July 2015 Published: 21 August 2015
Journal Compilation © CSIRO Publishing 2017 Open Access CC BY-NC-ND
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.
Additional keywords: single blastocyst, normalisation, qRT-PCR.
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