Excellion Biomedical Services, Petrópolis, Rio de Janeiro, Brazil.
PLoS One. 2013 Sep 2;8(9):e73792. doi: 10.1371/journal.pone.0073792. eCollection 2013.
Quantitative real time polymerase chain reaction (qPCR) is an extremely powerful technique for monitoring gene expression. The quantity of the messenger ribonucleic acids (mRNA) of interest should be normalized using a reference gene, in order to avoid unreliable results originated by the obtained RNA quality and quantity, manipulation errors and inhibitory contaminants. A reference gene is any gene that is stably and consistently expressed under the conditions being studied. Completely false data can be generated if a reference gene is not chosen adequately.
In the present study, we compared expression levels of five putative reference genes (HPRT1, ACTB, GAPDH, RPL13A and B2M) in primary cultures of four different human cells: mesenchymal stromal cells obtained from bone marrow, adipose tissue or umbilical cord Whartońs Jelly, and dermal fibroblasts, under different expansion and differentiation conditions. We observed that reference genes are not the same for different cells under the same culture conditions.
Most stable reference genes under our experimental conditions were: RPL13A for adipose tissue- and Whartońs Jelly-derived mesenchymal stromal cells, and HPRT1 for bone marrow-derived mesenchymal stromal cells and dermal fibroblasts. ACTB was the most unstable gene when evaluating adipose tissue- and Whartońs Jelly-derived mesenchymal stromal cells, whilst GAPDH and B2M were the most unstable genes for bone marrow-derived mesenchymal stromal cells and dermal fibroblasts, respectively.
实时荧光定量聚合酶链反应（qPCR）是一种监测基因表达的极其强大的技术。为了避免由于获得的 RNA 质量和数量、操作误差和抑制性污染物引起的不可靠结果，应使用参考基因对感兴趣的信使核糖核酸（mRNA）的量进行标准化。参考基因是在研究条件下稳定且一致表达的任何基因。如果没有选择合适的参考基因，则可能会产生完全错误的数据。
在本研究中，我们比较了四种不同的人细胞（骨髓、脂肪组织或脐带 Wharton 胶来源的间充质基质细胞和真皮成纤维细胞）的原代培养物在不同的扩增和分化条件下，五种假定参考基因（HPRT1、ACTB、GAPDH、RPL13A 和 B2M）的表达水平。我们观察到，在相同的培养条件下，不同细胞的参考基因并不相同。
在我们的实验条件下最稳定的参考基因是：脂肪组织和 Wharton 胶来源的间充质基质细胞中的 RPL13A，以及骨髓来源的间充质基质细胞和真皮成纤维细胞中的 HPRT1。在评估脂肪组织和 Wharton 胶来源的间充质基质细胞时，ACTB 是最不稳定的基因，而 GAPDH 和 B2M 分别是骨髓来源的间充质基质细胞和真皮成纤维细胞中最不稳定的基因。
