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<p>Canine mesenchymal stem cells (cMSCs) are increasingly utilized in veterinary regenerative medicine due to their immunomodulation functions, self-renewal capacity, and multi-lineage differentiation potential. Accurate gene expression analysis using quantitative real-time polymerase chain reaction (RT-qPCR) is essential for understanding cMSCs biology and therapeutic function; however, its reliability critically depends on normalization using stably expressed reference genes (RGs), which can vary under different experimental conditions. Because MSC phenotype and gene expression profiles can vary depending on tissue origin, replicative stages during in vitro expansion, and differentiation status, selecting appropriate RGs for each condition is essential. Despite growing interest of cMSCs, comprehensive validation of RG stability across these various conditions has not been reported. In this study, we evaluated the expression stability of ten commonly used RGs (ACTB, GAPDH, HPRT1, B2M, SDHA, TBP, GUSB, RPL4, RPS5, and YWHAZ) in cMSCs derived from four tissue sources (adipose, bone marrow, synovial fluid, and skin), across replicative stages of in vitro expansion (early, mid, and late passages), and following tri-lineage differentiation (adipogenic, osteogenic, and chondrogenic). Gene stability was assessed using two algorithms, geNorm and NormFinder. HPRT1 was consistently identified as the most stable RG across all tested conditions, whereas commonly used genes such as GAPDH and B2M showed low stability. Overall, these findings identify HPRT1 as a suitable reference gene for RT-qPCR analysis in cMSCs under diverse experimental conditions. These findings provide the first comprehensive validation of RG stability in cMSCs and highlight the importance of optimizing normalization strategies for future research and clinical applications.</p>

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Selection of stable reference genes for accurate RT-qPCR analysis in canine MSCs across tissues, replicative stages, and differentiation states

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年8月5日

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  • Chan-Hee Jo,
  • Chae-Yeon Hong,
  • Sang-Yun Lee,
  • Eun-Yeong Bok,
  • Young-Bum Son,
  • Won-Jae Lee,
  • Seong-Ju Oh,
  • Tae-Seok Kim,
  • Seo-Yoon Kang,
  • Sanghyeon Park,
  • Sung-Lim Lee,
  • Yong-ho Choe

摘要

Canine mesenchymal stem cells (cMSCs) are increasingly utilized in veterinary regenerative medicine due to their immunomodulation functions, self-renewal capacity, and multi-lineage differentiation potential. Accurate gene expression analysis using quantitative real-time polymerase chain reaction (RT-qPCR) is essential for understanding cMSCs biology and therapeutic function; however, its reliability critically depends on normalization using stably expressed reference genes (RGs), which can vary under different experimental conditions. Because MSC phenotype and gene expression profiles can vary depending on tissue origin, replicative stages during in vitro expansion, and differentiation status, selecting appropriate RGs for each condition is essential. Despite growing interest of cMSCs, comprehensive validation of RG stability across these various conditions has not been reported. In this study, we evaluated the expression stability of ten commonly used RGs (ACTB, GAPDH, HPRT1, B2M, SDHA, TBP, GUSB, RPL4, RPS5, and YWHAZ) in cMSCs derived from four tissue sources (adipose, bone marrow, synovial fluid, and skin), across replicative stages of in vitro expansion (early, mid, and late passages), and following tri-lineage differentiation (adipogenic, osteogenic, and chondrogenic). Gene stability was assessed using two algorithms, geNorm and NormFinder. HPRT1 was consistently identified as the most stable RG across all tested conditions, whereas commonly used genes such as GAPDH and B2M showed low stability. Overall, these findings identify HPRT1 as a suitable reference gene for RT-qPCR analysis in cMSCs under diverse experimental conditions. These findings provide the first comprehensive validation of RG stability in cMSCs and highlight the importance of optimizing normalization strategies for future research and clinical applications.