School of Materials Science and Engineering, South China University of Technology, Guangzhou, Guangdong, China.
PLoS One. 2013 May 21;8(5):e64786. doi: 10.1371/journal.pone.0064786. Print 2013.
Real-time quantitative reverse transcription-polymerase chain reaction (qPCR) is an efficient and accurate method to detect and compare patterns of gene expression. The reliability of qPCR is highly dependent on the selection of appropriate reference genes used for normalization. By analyzing 16 potential candidates of reference genes (GAPDH, Actb, 18 s, PGK1, Hprt, Tbp, Rpl5, B2M, Gusb, Ppia, UBC, Sdha, Eef1a1, H2afz, Tkt and Ldha) through geNorm, we identified Ppia, Tbp, Hprt and Eef1a1 as the most stable reference genes while UBC, B2M, Gusb as the least stable ones during the chondrocyte differentiation of ATDC5 cells. Considering the low expression of Eef1a1 and Tbp would cause divergent results for they failed to provide accurate normalization for RNA extraction and reverse transcription efficiency, we recommended the use of Ppia and Hprt as the most suitable genes to normalize qPCR. In addition, although GAPDH, Actb and 18 s were usually adopted in most of studies using ATDC5 cells, they were found unstable and then were not ideal reference genes for qPCR assay in ATDC5 cells chondrocyte differentiation. Also, we further confirmed that the Ppia and Hprt worked well during chondrocyte differentiation of mouse mesenchymal cells.
实时荧光定量逆转录聚合酶链反应（qPCR）是一种高效、准确的检测和比较基因表达模式的方法。qPCR 的可靠性高度依赖于用于标准化的合适参考基因的选择。通过对 16 个潜在的参考基因候选物（GAPDH、Actb、18s、PGK1、Hprt、Tbp、Rpl5、B2M、Gusb、Ppia、UBC、Sdha、Eef1a1、H2afz、Tkt 和 Ldha）进行 geNorm 分析，我们确定了 Ppia、Tbp、Hprt 和 Eef1a1 是 ATDC5 细胞软骨分化过程中最稳定的参考基因，而 UBC、B2M 和 Gusb 是最不稳定的参考基因。考虑到 Eef1a1 和 Tbp 的低表达会导致结果不一致，因为它们不能为 RNA 提取和反转录效率提供准确的标准化，我们建议使用 Ppia 和 Hprt 作为最适合的基因来标准化 qPCR。此外，尽管 GAPDH、Actb 和 18s 在大多数使用 ATDC5 细胞的研究中通常被采用，但我们发现它们不稳定，因此不是 ATDC5 细胞软骨分化中 qPCR 检测的理想参考基因。此外，我们还进一步证实，在小鼠间充质细胞的软骨分化过程中，Ppia 和 Hprt 效果良好。
