1Division of Cell Metabolism and Differentiation Research, Institute for Biomedical Aging Research, University of Innsbruck, Rennweg 10, A-6020 Innsbruck, Austria.
2Department of Plastic and Reconstructive Surgery, Innsbruck Medical University, Anichstraße 35, A-6020 Innsbruck, Austria.
Cell Mol Biol Lett. 2019 Feb 15;24:14. doi: 10.1186/s11658-019-0140-6. eCollection 2019.
The proliferation and adipogenic differentiation of adipose stromal cells (ASCs) are complex processes comprising major phenotypical alterations driven by up- and downregulation of hundreds of genes. Quantitative RT-PCR can be employed to measure relative changes in the expression of a gene of interest. This approach requires constitutively expressed reference genes for normalization to counteract inter-sample variations due to differences in RNA quality and quantity. Thus, a careful validation of quantitative RT-PCR reference genes is needed to accurately measure fluctuations in the expression of genes. Here, we evaluated candidate reference genes applicable for quantitative RT-PCR analysis of gene expression during proliferation and adipogenesis of human ASCs with the immunophenotype DLK1/CD34/CD90/CD105/CD45/CD31.
We evaluated the applicability of 10 candidate reference genes (, , , , , , , , and ) using NormFinder, geNorm and BestKeeper software.
The results indicate that and are the most reliable reference genes for quantitative RT-PCR analysis of proliferating ASCs. serves as the most reliable endogenous control in adipogenesis. and were among the least consistent genes.
Applying these findings for future gene expression analyses will help elucidate ASC biology.
脂肪基质细胞（ASCs）的增殖和脂肪分化是一个复杂的过程，包括由数百个基因的上调和下调驱动的主要表型改变。实时定量 RT-PCR 可用于测量目的基因表达的相对变化。这种方法需要使用恒表达的参考基因进行归一化，以抵消由于 RNA 质量和数量的差异导致的样品间差异。因此，需要仔细验证实时定量 RT-PCR 参考基因，以准确测量基因表达的波动。在这里，我们评估了候选参考基因在具有免疫表型 DLK1/CD34/CD90/CD105/CD45/CD31 的人 ASC 增殖和脂肪生成过程中进行实时定量 RT-PCR 分析时的适用性。
我们使用 NormFinder、geNorm 和 BestKeeper 软件评估了 10 个候选参考基因（、、、、、、、、和）的适用性。
结果表明，和是用于分析增殖 ASC 的实时定量 RT-PCR 分析最可靠的参考基因。在脂肪生成中，作为最可靠的内参基因。和是最不一致的基因之一。
将这些发现应用于未来的基因表达分析将有助于阐明 ASC 生物学。
