Validation of Optimal Reference Genes for qRT-PCR in Adipose- and Uterine-Derived Feline Mesenchymal Stem Cells

Feline mesenchymal stem cells (MSCs) isolated from adipose (A-MSCs) and uterine (U-MSCs) tissues during routine ovariohysterectomy represent valuable therapeutic sources in veterinary regenerative medicine. While quantitative real-time PCR (qRT-PCR) remains the gold standard for transcriptional profiling in these cells, its analytical reliability relies on empirically validated reference genes. In this study, we assessed the expression stability of nine candidate reference genes (ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, RPL7, TBP, and YWHAZ) in feline A-MSCs and U-MSCs using the geNorm and NormFinder algorithms. Both computational approaches identified TBP and HMBS as the most stably expressed genes across both cell types, whereas the historically popular GAPDH was the least stable. All geNorm pairwise variation values were below the commonly used threshold of 0.15 in both A-MSCs and U-MSCs. To illustrate the impact of reference gene choice, we evaluated the pluripotency marker OCT4. Normalizing using TBP and HMBS revealed significant differences in OCT4 expression between A-MSCs and U-MSCs (p < 0.05). In contrast, applying the unstable GAPDH masked this difference. These findings identify TBP and HMBS as promising reference gene candidates for qRT-PCR normalization in P3 undifferentiated feline A-MSCs and U-MSCs.