Tissue Engineering, Biomaterials and Nanobiotechnology Laboratory, Ankara University Faculty of Science, and Ankara University Stem Cell Institute, Ankara, Turkey.
Biovalda Health Technologies, Inc., Ankara, Turkey.
Mol Biol Rep. 2020 Mar;47(3):2109-2122. doi: 10.1007/s11033-020-05311-y. Epub 2020 Feb 17.
Mesenchymal stem cells (MSCs) from a variety of sources are being used in pre-clinical and clinical studies. The choice of optimal source for treatment of diseases requires quantitative evaluation of self-renewal, proliferation and differentiation potencies of MSCs. For this purpose, quantitative real-time polymerase chain reaction (qRT-PCR) technique is used to determine the expression of genes. qRT-PCR requires the normalization of the gene expression levels by the use of reference genes in order to obtain accurate and reliable results. There is a limited number of studies focused on the selection of reference genes that are appropriate and reliable for MSCs. Thus, no single reference gene has yet been found for use in the in vitro proliferation and differentiation of MSCs. The aim of this study is to investigate the stability of the expression of widely used reference genes during the in vitro proliferation and differentiation of human adipose-derived mesenchymal stem cells (hASCs). For this purpose, 13 reference genes commonly used in MSC studies were selected. As a result, the expression stabilities of EF1α, RPLP0 and RPL13A genes were found to be high and were predicted to be suitable for use as reference genes for normalization in hASC studies. The GAPDH was identified as the gene with the lowest expression stability and evaluated to be an unsuitable reference gene for hASC differentiation studies. This piece of information could be crucial for the selection of appropriate reference genes and accurate measurement of gene expression in hASC studies.
间充质干细胞（MSCs）来源于多种不同的组织，目前被广泛应用于临床前和临床试验中。为了治疗疾病，选择最佳的来源，需要对 MSCs 的自我更新、增殖和分化潜能进行定量评估。为此，人们采用实时定量聚合酶链反应（qRT-PCR）技术来测定基因的表达。qRT-PCR 需要通过使用参考基因对基因表达水平进行归一化，才能获得准确可靠的结果。然而，目前仅有少量研究关注适合和可靠的 MSCs 参考基因的选择。因此，尚未找到适用于 MSCs 体外增殖和分化的单一参考基因。本研究旨在探讨在人脂肪间充质干细胞（hASC）体外增殖和分化过程中，广泛使用的参考基因表达的稳定性。为此，我们选择了 13 种常用于 MSC 研究的参考基因。结果表明，EF1α、RPLP0 和 RPL13A 基因的表达稳定性较高，适合作为 hASC 研究中归一化的参考基因。GAPDH 基因被鉴定为表达稳定性最低的基因，不适合用于 hASC 分化研究。这些信息对于选择合适的参考基因和准确测量 hASC 研究中的基因表达至关重要。
