Beer Lucian, Mlitz Veronika, Gschwandtner Maria, Berger Tanja, Narzt Marie-Sophie, Gruber Florian, Brunner Patrick M, Tschachler Erwin, Mildner Michael
Research Division of Biology and Pathobiology of the Skin, Department of Dermatology, Medical University of Vienna, Vienna, Austria.
Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Vienna, Austria.
Exp Dermatol. 2015 Oct;24(10):742-7. doi: 10.1111/exd.12759. Epub 2015 Jun 4.
Reverse transcription polymerase chain reaction (qRT-PCR) has become a mainstay in many areas of skin research. To enable quantitative analysis, it is necessary to analyse expression of reference genes (RGs) for normalization of target gene expression. The selection of reliable RGs therefore has an important impact on the experimental outcome. In this study, we aimed to identify and validate the best suited RGs for qRT-PCR in human primary keratinocytes (KCs) over a broad range of experimental conditions using the novel bioinformatics tool 'RefGenes', which is based on a manually curated database of published microarray data. Expression of 6 RGs identified by RefGenes software and 12 commonly used RGs were validated by qRT-PCR. We assessed whether these 18 markers fulfilled the requirements for a valid RG by the comprehensive ranking of four bioinformatics tools and the coefficient of variation (CV). In an overall ranking, we found GUSB to be the most stably expressed RG, whereas the expression values of the commonly used RGs, GAPDH and B2M were significantly affected by varying experimental conditions. Our results identify RefGenes as a powerful tool for the identification of valid RGs and suggest GUSB as the most reliable RG for KCs.
逆转录聚合酶链反应（qRT-PCR）已成为皮肤研究许多领域的主要手段。为了进行定量分析，有必要分析参考基因（RGs）的表达以对靶基因表达进行标准化。因此，可靠的RGs的选择对实验结果有重要影响。在本研究中，我们旨在使用新型生物信息学工具“RefGenes”，在广泛的实验条件下，鉴定和验证最适合用于人原代角质形成细胞（KCs）qRT-PCR的RGs，该工具基于一个人工整理的已发表微阵列数据数据库。通过qRT-PCR验证了RefGenes软件鉴定的6个RGs和12个常用RGs的表达。我们通过四种生物信息学工具的综合排名和变异系数（CV）评估了这18个标志物是否满足有效RG的要求。在总体排名中，我们发现GUSB是表达最稳定的RG，而常用的RGs GAPDH和B2M的表达值受不同实验条件的显著影响。我们的结果表明RefGenes是鉴定有效RGs的有力工具，并表明GUSB是KCs最可靠的RG。
