Recommendations for improving accuracy of gene expression data in bone and cartilage tissue engineering.

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.