TBP and HPRT1 are the Most Suitable Reference Genes for Normalization of mRNA Expression in Feline Gonadal Tissue-Derived Mesenchymal Stem Cells

초록

Mesenchymal stem cells (MSCs), especially those derived from gonadal tissues that are easy to collect during routine sterilization procedures, provide an ethically viable source of feline MSCs. Considering MSCs are potential candidates for regenerative medicine, reporting therapeutic effects suggests that further research to develop more effective therapy using feline gonadal MSCs is needed for clinical application. Gene expression analysis and therapeutic potential of MSCs can be analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Reliable interpretation of results requires accurate normalization. Hence, this study focused on determining the highest stability of candidate reference genes and accurate gene expression determination in ovary-derived MSCs (O-MSCs) and testis-derived MSCs (T-MSCs). O-MSCs and T-MSCs were retrieved from three female and three male felines, and then their stemness properties, which included spindle-like morphology, specific surface markers, and trilineage differentiation capacity, were characterized. To rank the nine candidate reference gene stability and pairwise variation stability, geNorm and NormFinder were used. In terms of overall analysis, TBP and HPRT1 had the lowest stability values (<0.03), whereas GUSB, RPL7, and ACTB had higher stability values (>0.04). When stable reference genes were utilized for normalization, statistically significant differences in OCT4 expression were observed between O-MSCs and T-MSCs (P<0.05), which were not observed with unstable reference genes. This provides a methodological framework by which future qRT-PCR-based studies can be conducted on feline gonadal MSCs. The potential therapeutic applications of these cells can also be studied.

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