Identification of Most Stably Expressed Internal Control Genes for Normalization of Target Gene Expression in Buffalo Skin Tissue

Water buffalo (Bubalus bubalis) skin exhibits unique morphological and physiological adaptations compared to other domestic bovines to enhance thermoregulation facilitated by abundant dermal melanin. Melanin synthesis and distribution, governed by the melanogenesis pathway and associated genes underpin coat color phenotypes and determine thermotolerance. The molecular mechanisms regulating pigmentation in buffalo skin remain underexplored as well as the reliable internal control genes (ICGs) for qPCR normalization. This study addresses the critical gap by systematically evaluating a panel of nine candidate reference genes for their expression stability in skin tissues from contrastingly pigmented buffaloes, jet-black Murrah and Nili Ravi (CC1) versus extensively white-spotted Nili Ravi (CC2). From the application of comprehensive statistical algorithms (deltaC_q, NormFinder, geNorm, BestKeeper), the combination of GAPDH, EEF1A1, and RPS23 emerged as the most stable reference gene set for normalization. Validation using key melanogenesis genes (KIT and TYR) confirmed that normalization with this trio yields consistent and biologically relevant expression profiles, with KIT robustly downregulated in white versus black skin. The recommended reference gene combination ensures robust normalization in gene expression profiling of melanogenesis-related genes in buffalo skin tissue.