Housekeeping gene stability influences the quantification of osteogenic markers during stem cell differentiation to the osteogenic lineage
Housekeeping gene stability influences the quantification of osteogenic markers during stem cell differentiation to the osteogenic lineage
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DOI：
10.1007/s10616-010-9265-1
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发表时间：
2010-04-01
期刊：
影响因子：
2.2
通讯作者：
Lopez, Luis E.
中科院分区：
文献类型：
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作者：
Garcia Quiroz, Felipe;Posada, Olga M.;Lopez, Luis E.
实时逆转录PCR（RT-qPCR）依赖于管家基因或标准化基因，其表达在整个实验中保持恒定。RT-qPCR通常用于表征人骨髓间充质干细胞（hBMSC）。然而，据我们所知，没有研究证实在hBMSC分化过程中用作标准化物的基因的表达稳定性。本研究旨在研究hBMSCs成骨分化过程中管家基因β-肌动蛋白、甘油醛-3-磷酸脱氢酶（GAPDH）和核糖体蛋白L13 A（RPL 13 A）的稳定性。通过RT-qPCR在14天和20天的成骨谱系分化测定中评价其稳定性。不同的标准化策略进行了评估，以量化成骨标志物胶原蛋白I型，骨唾液酸蛋白和骨粘连蛋白。通过茜素红染色证实细胞分化。结果表明β-肌动蛋白上调，最大倍数变化（MFC）为4.38。14天后，GAPDH和RPL 13 A不再受到成骨培养基的调节，并且在20天的培养物中平均倍数变化低于2。与GAPDH（MFC B 2.2）相比，RPL 13 A（MFC < 2）在作为培养时间的函数归一化时具有更大的稳定性，这导致成骨标志物的表达模式与观察到的分化过程更一致。结果表明，β-actin的调控可能与hBMSCs成骨分化的形态学变化特征有关，并为RPL 13 A作为正常化基因在hBMSCs成骨分化研究中的上级性能提供了证据。这项工作强调了通过RT-qPCR验证用于干细胞表征的标准化基因的重要性。
Real-time reverse transcription PCR (RT-qPCR) relies on a housekeeping or normalizer gene whose expression remains constant throughout the experiment. RT-qPCR is commonly used for characterization of human bone marrow mesenchymal stem cells (hBMSCs). However, to the best of our knowledge, there are no studies validating the expression stability of the genes used as normalizers during hBMSCs differentiation. This work aimed to study the stability of the housekeeping genes beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal protein L13A (RPL13A) during the osteogenic differentiation of hBMSCs. Their stability was evaluated via RT-qPCR in 14 and 20 day differentiation assays to the osteogenic lineage. Different normalization strategies were evaluated to quantify the osteogenic markers collagen type I, bone sialoprotein and osteonectin. Cell differentiation was confirmed via alizarin red staining. The results demonstrated up-regulation of beta-actin with maximum fold changes (MFC) of 4.38. GAPDH and RPL13A were not regulated by osteogenic media after 14 days and presented average fold changes lower than 2 in 20 day cultures. RPL13A (MFC < 2) had a greater stability when normalizing as a function of culture time compared with GAPDH ( MFC B 2.2), which resulted in expression patterns of the osteogenic markers more consistent with the observed differentiation process. The results suggest that beta-actin regulation could be associated with the morphological changes characteristic of hBMSCs osteogenic differentiation, and provide evidence for the superior performance of RPL13A as a normalizer gene in osteogenic differentiation studies of hBMSCs. This work highlights the importance of validating the normalizer genes used for stem cells characterization via RT-qPCR.