Selection and Expression Profiles of Reference Genes in Mouse Preimplantation Embryos of Different Ploidies at Various Developmental Stages

Abstract

Real-time reverse transcription quantitative polymerase chain reaction (qPCR) has become the most frequently used system for studies of gene expression. Manystudies have provided reliable evidence that the transcription levels of reference genes are not constant at different developmental stages and in different experimental conditions. However, suitable reference genes which are stably expressed in polyploid preimplantation embryos of different developmental stages have not yet been identified. Therefore, it is critical to verify candidate reference genes to analyze gene expression accurately in both diploid and polyploid embryos. We examined the expression levels of 12 candidate reference genes in preimplantation embryos of four different ploidies at six developmental stages. Stability analysis of the reference genes was performed by four independent software programs, and the stability of three genes was evaluated by comparison with the Oct4 expression level during preimplantation development in diploid embryos. The expression levels of most genes in the polyploid embryos were higher than that in the diploid embryos, but the increasing degree were disproportionate with the ploidies. There were no significant difference in reference gene expressions among embryos of different ploidies when they reached the morula stage, and the expression level remained flat until the blastocyst stage. Ubc, Ppia, and Pgk1 were the three most stable reference genes in diploid and polyploid embryos.

Selection of reference genes for quantitative real-time polymerase chain reaction in porcine embryos

“…Several previous studies have provided evidence that transcription levels of reference genes from embryos did not express constantly in different developmental stages and under different production methods (Mamo et al 2007(Mamo et al , 2008Smits et al 2009;Gu et al 2014). In the present study, the differences in CT values of reference gene expression were observed in blastocysts produced by different in vivo and in vitro methods (Fig.…”

“…Furthermore, an investigation focussed on the inefficiency of porcine SCNT embryos has revealed that abnormal patterns of developmental efficiency and gene expression were caused by incomplete reprogramming of the donor cell genome as well as aberrant epigenetic mechanisms related to DNA methylation and histone de-acetylation (Kumar et al 2007(Kumar et al , 2013Zhao et al Relative expressions of OCT4 2010). It has been reported that reference genes in early embryonic developmental stages were differentially expressed and hence the accurate assessment of gene expression could not be performed (Robert et al 2002;Mamo et al 2007Mamo et al , 2008Gu et al 2014). Gene expression of different embryos reflects their own characteristics, therefore, investigations should be focussed on increasing the accuracy of reliable reference genes for appropriate comparative gene expression studies.…”

“…Accordingly, even if some reference genes could be evaluated as having stable expression in certain embryos, there have been different results depending upon different species. Several studies have been performed to answer this issue of validating stable reference genes in embryos of different species, including mouse (Jeong et al 2005;Mamo et al 2007;Llobat et al 2012;Gu et al 2014), cow (Robert et al 2002;Goossens et al 2005;Luchsinger et al 2014), rabbit (Mamo et al 2008), horse (Smits et al 2009;Paris et al 2011) and chicken (Yang et al 2013). PPIA, H2A and HPRT1 were determined as the most-stable reference genes in mouse embryos produced in vivo and in vitro by geNorm analysis (Mamo et al 2007;Gu et al 2014).…”

“…Candidates of reference genes were selected on the basis of previously described articles examining the stability of porcine reference genes under several experimental conditions (Nygard et al 2007;Uddin et al 2011;McCulloch et al 2012;Lee et al 2015) and evaluated according to cases where they were applied as embryo reference genes (Goossens et al 2005;Bettegowda et al 2006;Mamo et al 2007;Smits et al 2009;Gu et al 2014;Luchsinger et al 2014). Consequently, nine widely used reference genes including 18S, H2A histone family, member Z (H2A), hypoxanthine phosphoribosyltransferase1 (HPRT1), GAPDH, ribosomal protein 4 (RPL4), peptidylprolyl isomerase A (PPIA), ACTB, succinate dehydrogenase complex, subunit A (SDHA) and hydroxymethylbilane synthase (HMBS2), were selected and sequences of all reference genes were taken from Nygard et al (2007) or Lee et al (2015).…”

“…Several studies have focussed on evaluation of reference genes from pre-implantation embryos in various species including mouse (Mamo et al 2007;Llobat et al 2012;Gu et al 2014), rabbit (Mamo et al 2008), horse (Smits et al 2009;Paris et al 2011) and cattle (Goossens et al 2005;Luchsinger et al 2014). Although several studies of porcine reference genes have been investigated under various conditions such as different tissues, age and treatments (Nygard et al 2007;Uddin et al 2011;McCulloch et al 2012), information on suitable reference genes from porcine embryos is available only from IVF embryos (Kuijk et al 2007).…”