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

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A Correction to this article was published on 30 October 2023

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Abstract

Background

Normalization 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 results

Six 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 ΔCt 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.

Conclusion

Our 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.

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Acknowledgements

Authors appreciate the help from other colleagues in Dr. Yang’s and Dr. Du’s groups.

Funding

Author Cai-Xia Yang and Zhi-Qiang Du have received Startup Grants from Yangtze University, China.

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Authors and Affiliations

  1. College of Animal Science, Yangtze University, 434025, Jingzhou, Hubei, China

    Yi Wang, Yu-Qing Zhang, Zi-Wei Wu, Ting Fang, Fang Wang, Han Zhao, Zhi-Qiang Du & Cai-Xia Yang

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  1. Yi Wang
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Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Yi Wang and Yu-Qing Zhang. Zi-Wei Wu, Ting Fang, Fang Wang and Han Zhao provided the helps for sample collection and figure organization. The first draft of the manuscript was written by Cai-Xia Yang and Zhi-Qiang Du edited the manuscript. All authors read and approved the final manuscript.

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Correspondence to Zhi-Qiang Du or Cai-Xia Yang.

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The authors have no relevant financial or non-financial interests to disclose.

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All experimental materials and procedures were reviewed and approved by the Animal Care Commission and Ethics Committee of the Yangtze University (YZU-2018-0031), Hubei, China.

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The original online version of this article was revised: The 25th reference “Ren J, Zhang N, Li X, Sun X, Song J (2021) Identification of reference genes for gene expression studies among different developmental stages of murine hearts. BMC Dev Biol 21(1):13. https://doi.org/10.1186/s12861-021-00244-6” is replaced with “Xie F, Xiao P, Chen D, Xu L, Zhang B. miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs. Plant Mol Biol. 2012;80:75. doi: 10.1007/s11103-012-9885-2.”

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Wang, Y., Zhang, YQ., Wu, ZW. et al. Selection of reference genes for RT-qPCR analysis in developing chicken embryonic ovary. Mol Biol Rep 50, 3379–3387 (2023). https://doi.org/10.1007/s11033-023-08280-0

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