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Screening and stability analysis of reference genes in fasting caecotrophy model in rabbits

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Abstract

Background

The selection and validation of stably expressed reference genes is key for accurately quantifying the mRNA abundance of genes under different treatments. In the rabbit model of fasting caecotrophy, reports about the selection of stable reference genes are not available.

Methods and results

This study aims to screen suitable reference genes in different tissues (including uterus, cecum, and liver) of rabbits between control and fasting caecotrophy groups. RT-qPCR was used to analyze the expression levels of eight commonly used reference genes (including GAPDH, 18S rRNA, B2M, CYP, HPRT1, β-actin, H2afz, Ywhaz), and RefFinder (including geNorm, NormFinder, and BestKeeper) was used to analyze the expression stability of these reference genes. Our results showed that the most stable reference genes were different in different tissues and treatments. In the control and fasting caecotrophy groups, CYP, GAPDH and HPRT1 were proven to be the top stable reference genes in the uterus, cecum, and liver tissues, respectively. GAPDH and Ywhaz were proven to be the top two stable reference genes among uterus, cecum, and liver in both control and fasting caecotrophy groups.

Conclusions

Our results indicated that the combined analysis of three or more reference genes (GAPDH, HPRT1, and Ywhaz) are recommended to be used for RT-qPCR normalization in the rabbit model of fasting caecotrophy, and that GAPDH is a better choice than the other reference genes for normalizing the relative expression of target genes in different tissues of fasting caecotrophy rabbits.

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Funding

This research was jointly supported by the “National Key Research and Development Program of China (2018YFD0502203)” and “the Special Fund for Henan Agriculture Research System (S2013-08-G01)”.

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Author notes

  1. Hui He and Zhichao Li have contributed equally to this paper.

Authors and Affiliations

  1. College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, 450046, People’s Republic of China

    Hui He, Zhichao Li, Mengke Ni, Shanshan Xing, Lei Yu, Huifen Xu & Ming Li

  2. College of Medicine, Zhengzhou University, Zhengzhou, 450046, People’s Republic of China

    Dehu Zhuo

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  1. Hui He
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Contributions

onceptualization, Huifen Xu, Ming Li and Hui He; funding acquisition, Ming Li; supervision, Mengke Ni; formal analysis, Shanshan Xing; validation, Zhichao Li and Lei Yu; writing-original draft, Hui He and Zhichao Li; writing-review and editing, Huifen Xu and Dehu Zhuo. All authors approved the final manuscript.

Corresponding authors

Correspondence to Huifen Xu, Dehu Zhuo or Ming Li.

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Conflict of interest

The authors declare no competing financial interest.

Ethical approval

This study was designed and performed according to the guidelines of the institutional Animal Care and Use Committee (IACUC) of College of Animal Science and technology of Henan Agricultural University, China (No: 11-0085).

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He, H., Li, Z., Ni, M. et al. Screening and stability analysis of reference genes in fasting caecotrophy model in rabbits. Mol Biol Rep 49, 1057–1065 (2022). https://doi.org/10.1007/s11033-021-06927-4

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