Dipartimento Scienze Cliniche Specialistiche ed Odotontostomatologiche, Università Politecnica delle Marche, I-60131 Ancona, Italy.
J Biotechnol. 2011 Dec 10;156(3):153-62. doi: 10.1016/j.jbiotec.2011.09.018. Epub 2011 Sep 22.
Data normalization of gene expression on human dermal fibroblasts (HDF) exposed to UVA has commonly been done using either GAPDH or β-actin as reference genes without any validation of their expression stability. Since this aspect, important for accurate normalization, has been overlooked, we aimed to establish a suitable set of reference genes for studies on UVA-treated HDF cultured under both standard atmospheric oxygen tension (normoxia, 21%) and under a physiological, low oxygen tension for these cells (hypoxia, 5%). The stability of six commonly used reference genes was assessed using the geNorm and NormFinder softwares subsequent to reverse-transcription quantitative real-time PCR (RT-qPCR). GAPDH/SDHA were found to be the most stable genes under normoxia, while SDHA/TBP or HPRT1/β2M were the most stable ones under hypoxia in HDF exposed to 18 J/cm(2) UVA. β-Actin was always the most unstable reference gene. To emphasize the importance of selecting the most stably expressed reference genes for obtaining reliable results, mRNA expression levels of MMP-1 and COL1A1 were analyzed vs the best reference genes and the worst one. These reference genes are hence recommended for future qPCR analyses in studies concerning photo-damage on UVA-treated HDF.
已经普遍使用 GAPDH 或 β-肌动蛋白作为参照基因来对暴露于 UVA 的人真皮成纤维细胞（HDF）的基因表达进行数据归一化，而没有对其表达稳定性进行任何验证。由于这一方面对于准确的归一化很重要，但却被忽视了，因此我们旨在为在标准大气氧张力（常氧，21%）和细胞的生理低氧张力（缺氧，5%）下培养的 UVA 处理的 HDF 研究建立合适的参照基因集。使用 geNorm 和 NormFinder 软件对逆转录定量实时 PCR（RT-qPCR）后的六个常用参照基因的稳定性进行了评估。在常氧条件下，发现 GAPDH/SDHA 是最稳定的基因，而在暴露于 18 J/cm(2) UVA 的 HDF 中，SDHA/TBP 或 HPRT1/β2M 是最稳定的基因。β-肌动蛋白始终是最不稳定的参照基因。为了强调选择最稳定表达的参照基因以获得可靠结果的重要性，分析了 MMP-1 和 COL1A1 的 mRNA 表达水平与最佳参照基因和最差参照基因的关系。因此，建议在有关 UVA 处理的 HDF 光损伤的未来 qPCR 分析中使用这些参照基因。
