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Comprehensive evaluation and validation of optimal reference genes for normalization of qPCR data in different caprine tissues

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Abstract

Background

Quantitative real-time PCR (qPCR) is a highly reliable method for validating gene expression data in molecular studies due to its sensitivity, specificity, and efficiency. To ensure accurate qPCR results, it’s essential to normalize the expression data using stable reference genes.

Methods

This study aimed to identify suitable reference genes for qPCR studies in goats by evaluating 18 candidate reference genes (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) in 10 different caprine tissues (heart, intestine, kidney, liver, lung, muscle, rumen, skin, spleen, and testis). An integrated tool called RefFinder, which incorporates various algorithms like NormFinder, GeNorm, BestKeeper, and ΔCt, was used to assess the stability of expression among these genes.

Results

After thorough analysis, ACTB, PPIB, and B2M emerged as the most stable reference genes, while RPL19, RPS15, and RPS9 were found to be the least stable. The suitability of the selected internal control genes was further validated through target gene analysis, confirming their efficacy in ensuring accurate gene expression profiling in goats.

Conclusion

The study determined that the geometric average of ACTB, PPIB, and B2M creates an appropriate normalization factor for gene expression studies in goat tissues.

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Data availability

No datasets were generated or analysed during the current study.

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Funding

This work was financially supported by the ICAR-Consortium Research Platform- Genomics.

Author information

Authors and Affiliations

  1. ICAR-National Bureau of Animal Genetic Resources, Karnal, India

    Sonika Ahlawat, Mahanthi Vasu, Reena Arora & Rekha Sharma

  2. District Disease Diagnostic Laboratory, Department of Animal Husbandry and Dairying, Karnal, Haryana, India

    Vikas Choudhary

  3. Mountain Research Centre for Sheep and Goat, SKUAST, Shuhama (Aulestang), Kashmir, India

    MA Mir

  4. ICAR-Central Institute for Research on Goats, Makhdoom, Mathura, India

    Manoj Kumar Singh

  5. Animal Biotechnology Division, ICAR-NBAGR, Karnal, India

    Sonika Ahlawat

Authors
  1. Sonika Ahlawat
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  2. Mahanthi Vasu
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  3. Vikas Choudhary
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  4. Reena Arora
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  5. Rekha Sharma
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  6. MA Mir
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  7. Manoj Kumar Singh
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Contributions

S.A.: conceptualization, generation of the manuscript. M.V.: data curation, formal analyses. V.C., M.M., and M.K.S.: resources. R.A. and R.S.: formal analyses. All authors reviewed the manuscript.

Corresponding author

Correspondence to Sonika Ahlawat.

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Ethical approval

This work was approved by the Institutional Animal Ethics Committee (IAEC) of ICAR-National Bureau of Animal Genetics Resources (F. No. NBAGR/IAEC/2017, dated 21.01.2017).

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The authors declare no competing interests.

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Ahlawat, S., Vasu, M., Choudhary, V. et al. Comprehensive evaluation and validation of optimal reference genes for normalization of qPCR data in different caprine tissues. Mol Biol Rep 51, 268 (2024). https://doi.org/10.1007/s11033-024-09268-0

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