Abstract
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.
Highlights
After more than a century, clinical bone regenerative procedures still rely on autogenous bone grafting[1,2,3] to heal and regenerate large bone defects in human
For human bone-derived mesenchymal stem cells, the mean Cq of ribosomal protein L13a (RPL13a) was consistent with complementary DNA quantity ranging from 2.5 ng to 40 ng (20.03 ± 0.68); the mean Cq of SDHA, TATA-binding protein (TBP), RNA28S4, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and RPLP0 decreased as the amount of cDNA increased; the mean Cq of POLR2e and actin beta (ACTB) increased as the amount of cDNA increased (Table 1)
For the rat rectus abdominis muscle, the mean threshold cycle of TBP and GAPDH remained constant independently of the cDNA quantity utilized (26.46 ± 0.96, and 26.64 ± 0.24 respectively); the mean Cq of POLR2e and RNA28S4 decreased as the amount of cDNA increased for the muscle tissue with the mean Cq of SDHA, ACTB, RPL13a, and RPLP0 increasing as the amount of cDNA increased (Table 1)
Summary
After more than a century, clinical bone regenerative procedures still rely on autogenous bone grafting[1,2,3] to heal and regenerate large bone defects in human. Real-time reverse transcription quantitative polymerase chain reaction (qRT-PCR) has become the leading analytic technique, and is an economical, jet simple and highly effective method for monitoring gene transcription, which over the last two decades has made considerable progress at improving the accuracy of gene expression data[21,22,23,24,25,26,27,28,29,30,31]. Whilst some bone and cartilage research groups have already used the new standards as set out by Bustin et al.[27] pioneering “Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines”[20,27,32,33,34], a considerable number of research groups still rely on outdated and highly insufficient qRT-PCR techniques; when combined with insufficient experimental detail, these techniques render replication of many published findings challenging[35] and questionable. 5 ng preventing regenerative therapies from replacing the gold standards currently employed to treat bone and cartilage damage and defects
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