Quantitative evaluation and selection of reference genes in mouse oocytes and embryos cultured in vivo and in vitro

Abstract

Background: Real-time PCR is an efficient tool to measure transcripts and provide valuable quantitative information on gene expression of preimplantation stage embryos. Finding valid reference genes for normalization is essential to interpret the real-time PCR results accurately, and understand the biological dynamics during early development. The use of reference genes also known as housekeeping genes is the most widely applied approach. However, the different genes are not systematically compared, and as a result there is no uniformity between studies in selecting the reference gene. The goals of this study were to compare a wide selection of the most commonly used housekeeping genes in mouse oocytes and preimplantation stage embryos produced under different culture conditions, and select the best stable genes for normalization of gene expression data.

Results: Quantitative real time PCR method was used to evaluate 12 commonly used housekeeping genes (Actb, Gapdh, H2afz, Hprt, Ppia, Ubc, Eef1e1, Tubb4, Hist2h2aa1, Tbp, Bmp7, Polr2a) in multiple individual embryos representing six different developmental stages. The results were analysed, and stable genes were selected using the geNorm software. The expression pattern was almost similar despite differences in the culture system; however, the transcript levels were affected by culture conditions. The genes have showed various stabilities, and have been ranked accordingly.

Conclusion: Compared to earlier studies with similar objectives, we used a unique approach in analysing larger number of genes, comparing embryo samples derived in vivo or in vitro, analysing the expression in the early and late maternal to zygote transition periods separately, and using multiple individual embryos. Based on detailed quantification, pattern analyses and using the geNorm application, we found Ppia, H2afz and Hprt1 genes to be the most stable across the different stages and culture conditions, while Actb, the classical housekeeping gene, showed the least stability. We recommend the use of the geometric averages of those three genes for normalization in mouse preimplantation-stage gene expression studies.

Figure 1

Individual expression profiles of selected reference genes in the in vivo derived embryos. The expression level in a particular developmental stage was represented with in vitro produced blastocyst embryo equivalent values, to show the relative amount. Stages with different letters are significantly (P ≤ 0.05) different for the expression of the gene.

Figure 2

Individual expression profiles of selected reference genes in the in vitro produced embryos. The expression level in a particular developmental stage was represented with in vitro produced blastocyst embryo equivalent values, to show the relative amount. Stages with different letters are significantly (P ≤ 0.05) different for the expression of the gene.

Figure 3

Relative expression levels of different transcripts in the in vivo derived preimplantation-stage mouse embryos. The expression at the oocyte stage was taken as a reference to calculate the relative amounts in the different stages.

Figure 4

Relative expression levels of different transcripts in the in vitro produced preimplantation-stage mouse embryos. The expression at the oocyte stage was taken as a reference to calculate the relative amounts in the different stages.

Figure 5

Average gene expression stability values of the selected reference genes in different cultures as calculated by geNorm software and ranking made based on the relative stability values.