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.
Highlights
Gene expression studies in tissue or cell samples are dependent on the use of appropriate reference genes
We examined the expression levels of these 12 candidate reference genes in preimplantation embryos of four different ploidies at six developmental stages (1-cell, 1C; 2-cell, 2C; 4-cell, 4C; morula, Mo; early blastocyst, EB; and late blastocyst, LB)
The transcripts of Gapdh, Pgk1, Ywhaz, Ppib, and Tbp were decreased from the 1C to 4C stage, and the Ywhaz, Ppib, and Tbp messenger RNA (mRNA) levels showed a sharp decrease
Summary
Gene expression studies in tissue or cell samples are dependent on the use of appropriate reference genes. Some researchers have proposed assuming that reference genes which are expressed at a constant level in tissues or at all stages of development are unaffected by experimental treatments. To study gene expression in preimplantation embryos, the reference genes should be expressed stably at various developmental stages, and the variation in expression levels should be small, but not immutable. These levels should gradually increase as embryonic development progresses and with the increasing numbers of cells
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call