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Selection of suitable reference genes for normalization of RT-qPCR in three tissues of Northern bobwhite (Colinus virginianus) infected with eyeworm (Oxyspirura petrowi)

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Abstract

Background

The Northern bobwhite (Colinus virginianus) is an economically important, and popular game bird in North America. Northern bobwhites have experiencing declines of > 3.5% annually in recent decades due to several factors. The eyeworm Oxyspirura petrowi is a nematode parasite frequently found in the eyes of bobwhites. Although reported frequently in wild bobwhites, there is no research to understand the host-parasite mechanism. Hence, it is important to investigate mechanisms of eyeworm invasion and immune modulation in bobwhite. Cytokine gene expression using RT-PCR is widely used to identify the innate immune response of a host to an infection.

Methodology

In this study, we evaluated ten reference genes (HMBS, RPL19, RPL32, RPS7, RPS8, TATA, SDHA, YWHAZ, GAPDH, and ACTB) for their stability across three tissues (liver, spleen, and caecal tonsils) of control and O. petrowi infected Northern bobwhites. Primer efficiency and reference genes stability were assessed using GeNorm, NormFinder, and BestKeeper.

Results

Expression of these reference genes with respect to O. petrowi infection in bobwhites showed RPL32 and HMBS were the most stable genes in the liver, HMBS and SDHA were the most stable genes in the spleen, and HMBS and YWHAZ were equally stable reference genes in the caecal tonsils.

Conclusion

Based on the geometric mean of all three analyses, our results indicate that the combination of RPL32 and HMBS for the liver, HMBS and SDHA for the spleen, and YWHAZ and HMBS for caecal tonsils might be used as reference genes for normalization in gene expression investigations on Northern bobwhites.

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Data availability

Data are available from the author upon reasonable request.

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Funding

This research received funding and support from Park Cities Quail and the Rolling Plains Quail Research Foundation.

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Authors and Affiliations

  1. The Wildlife Toxicology Laboratory, Texas Tech University, Lubbock, TX, 79409-3290, USA

    Aravindan Kalyanasundaram, Brett J. Henry, Cassandra Henry, Jeremiah Leach & Ronald J. Kendall

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  1. Aravindan Kalyanasundaram
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  2. Brett J. Henry
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  3. Cassandra Henry
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Contributions

A.K., B.J.H., C.H., and R.J.K. contributed equally to experimental design. A.K and B.J.H. contributed equally to experimental work and analysis and J.Leach contributed to data analysis. A.K and J.L contributed equally to the first draft of the manuscript and all authors provided edits and comments on all versions. All authors have read and approved the final manuscript.

Corresponding author

Correspondence to Ronald J. Kendall.

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Ethical statement.

This experiment was approved by Texas Tech University Animal Care and Use Committee under protocols 18044-05 and 16071-08. All bobwhites were trapped and handled according to Texas Parks and Wildlife permit SRPT-0715-095.

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Kalyanasundaram, A., Henry, B.J., Henry, C. et al. Selection of suitable reference genes for normalization of RT-qPCR in three tissues of Northern bobwhite (Colinus virginianus) infected with eyeworm (Oxyspirura petrowi). Mol Biol Rep 51, 483 (2024). https://doi.org/10.1007/s11033-024-09401-z

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