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Identification of Most Stably Expressed Internal Control Genes for Normalization of Target Gene Expression in Buffalo
Ankita Gurao1, Anurag Kumar1, Namita Kumari1
1ICAR-National Bureau of Animal Genetic Resources, Karnal, Haryana, India.
Environmental and Molecular Mutagenesis
|November 6, 2025
Summary
Researchers identified stable reference genes for studying water buffalo skin pigmentation. The combination of GAPDH, EEF1A1, and RPS23 is recommended for accurate gene expression analysis in melanogenesis studies.
Area of Science:
- Animal Genetics
- Dermatology
- Molecular Biology
Background:
- Water buffalo skin has unique adaptations for thermoregulation, involving dermal melanin.
- Melanin synthesis and gene regulation influence coat color and thermotolerance, but molecular mechanisms are poorly understood.
- Reliable internal control genes (ICGs) for quantitative PCR (qPCR) normalization in buffalo skin are lacking.
Purpose of the Study:
- To systematically evaluate candidate reference genes for expression stability in water buffalo skin.
- To identify the most reliable ICGs for normalizing gene expression data in melanogenesis research.
- To provide a validated normalization strategy for studying pigmentation in buffalo skin.
Main Methods:
- Evaluated nine candidate reference genes in skin tissues from black (CC1) and white-spotted (CC2) Nili Ravi buffalo.
- Applied statistical algorithms including deltaCq, NormFinder, geNorm, and BestKeeper.
- Validated the chosen reference gene set using melanogenesis genes KIT and TYR.
Main Results:
- The gene combination GAPDH, EEF1A1, and RPS23 demonstrated the highest expression stability across different pigmentation phenotypes.
- Normalization with this trio yielded consistent and biologically relevant gene expression profiles.
- The KIT gene was confirmed to be significantly downregulated in white versus black buffalo skin.
Conclusions:
- GAPDH, EEF1A1, and RPS23 are recommended as a stable reference gene set for qPCR normalization in water buffalo skin.
- This validated normalization strategy ensures robust gene expression profiling of melanogenesis-related genes.
- The findings contribute to a better understanding of the molecular basis of pigmentation and thermotolerance in water buffalo.
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