Laboratory of Biomechanics and Experimental Orthopaedics, Department of Orthopaedic Surgery, Physical Medicine and Rehabilitation, University Hospital of Munich (LMU), Munich, Germany.
Department of Orthopaedics, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
Sci Rep. 2018 Oct 5;8(1):14874. doi: 10.1038/s41598-018-33242-z.
Autogenous tissue grafting remains the gold standard in the treatment of critical sized bone and certain cartilage defects, while the translation of tissue engineered osteogenesis or chondrogenesis from the lab bench into clinical practice, utilizing natural or synthetic biomimetic devices, remains challenging. One of the crucial underestimated reasons for non-translatability could be the imprecision and inconsistency of generated gene expression profiles, utilizing improperly optimized and standardized quantitative gene assays. Utilizing GeNorm for downstream qRT-PCR applications, the stability of reference genes in relation to optimal cDNA amounts was assessed on human bone marrow-derived mesenchymal and adipose-derived stem cells neat and made to differentiate into chondrocytes including normal human derived chondrocytes and muscle tissue from rats. Results showed that reference genes can vary substantially across separately and/or combined cell lines and/or tissue types including treatment parameters. The recommendations to all bone and cartilage tissue engineers utilizing qRT-PCR is not to assume that reference gene stability and quantity remain conserved across cell lines or tissue types but to always determine, for each new experiment, the stability and normalization quantity of reference genes anew.
自体组织移植仍然是治疗临界大小骨和某些软骨缺陷的金标准，而利用天然或合成仿生设备将组织工程成骨或软骨形成从实验室转化为临床实践仍然具有挑战性。基因表达谱生成不精确和不一致可能是不可转化性的一个关键被低估的原因，这是利用不当优化和标准化的定量基因检测方法造成的。利用 GeNorm 进行下游 qRT-PCR 应用，评估了在人类骨髓间充质和脂肪间充质干细胞中，与最佳 cDNA 量相关的参考基因的稳定性，这些细胞被诱导分化为软骨细胞，包括正常的人源性软骨细胞和来自大鼠的肌肉组织。结果表明，参考基因在单独和/或组合的细胞系和/或组织类型（包括处理参数）之间可能会有很大的差异。建议所有使用 qRT-PCR 的骨和软骨组织工程师不要假设参考基因的稳定性和数量在细胞系或组织类型之间保持不变，而是始终为每个新实验确定参考基因的稳定性和新的归一化数量。
