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

Abstract

Simple Summary Gene expression analysis is essential for evaluating feline mesenchymal stem cells (MSCs). While quantitative real-time PCR (qRT-PCR) is a standard tool for this purpose, its accuracy depends on selecting stable reference genes for data normalization. The routine use of traditional reference genes without tissue-specific validation introduces severe analytical bias. Therefore, we evaluated nine candidate reference genes in feline MSCs isolated from adipose and uterine tissues. HMBS and TBP were the most stably expressed genes across both cell populations. Conversely, GAPDH exhibited the highest variability. To illustrate the impact of reference gene selection, we demonstrated that normalizing target gene expression against an unvalidated control can mask true biological differences between these cells. In conclusion, HMBS and TBP were identified as the most stably expressed reference gene candidates among the nine genes for evaluating gene expression using qRT-PCR in P3 undifferentiated feline A-MSCs and U-MSCs under the normal culture conditions in this study.