Selection of reference genes for RT-qPCR analysis in developing chicken embryonic ovary

BackgroundNormalization of the expression profiling of target genes, in a tissue-specific manner and under different experimental conditions, requires stably expressed gene(s) to be used as internal reference(s). However, to study the molecular regulation of oocyte meiosis initiation during ovary development in chicken embryos, stable reference gene(s) still need to be compared and confirmed.Methods and resultsSix candidate genes previously used as internal references for the chicken embryo (Actb, Cvh, Dazl, Eef1a, Gapdh and Rpl15) were chosen, and their expression profiles in left ovaries dissected at five chicken embryonic days (E12.5, E15.5, E17.5, E18.5 and E20.5) were evaluated, respectively. Separately, GeNorm, NormFinder, BestKeeper and Comparative Delta Ct methods were used to assess the stability of candidate reference genes, and all results were combined to give the final rank by RefFinder. All methods identified that Eef1a and Rpl15 were the two most stable internal reference genes, whereas Cvh is the most unstable one. Moreover, expression levels of three marker genes for chicken oocyte meiosis entry (Stra8, Scp3 and Dmc1) were normalized, based on Eef1a, Rpl15, or their combinations, respectively.ConclusionOur findings provide the most suitable internal reference genes (Eef1a and Rpl15), to investigate further molecular regulation of ovary development and oocyte meiosis initiation in chicken embryos.

Automated summary

This study evaluated the expression profiles of six candidate genes as internal reference genes in the ovaries of chicken embryos at different developmental stages. The most stable internal reference genes were identified as Eef1a and Rpl15, while Cvh was the least stable. Furthermore, the expression levels of three marker genes for oocyte meiosis entry were normalized using Eef1a, Rpl15, or their combinations.