Bioinformatics approach for choosing the correct reference genes when studying gene expression in human keratinocytes.

Lucian Beer, Veronika Mlitz, Maria Gschwandtner, Tanja Berger, Marie-Sophie Narzt, Florian Gruber, Patrick M Brunner, Erwin Tschachler, Michael Mildner

Journal: Experimental dermatology 2016;24(10):742-7

PMID: 25980460

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Abstract

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.

Address: 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.; Christian Doppler Laboratory for Biotechnology of Skin Aging, Department of Dermatology, Medical University of Vienna, Vienna, Austria.; Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Vienna, Austria.

DOI: 10.1111/exd.12759

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MeSH Terms: Actins, Biomarkers, Biomarkers, Tumor, Cell Differentiation, Computational Biology, Databases, Genetic, Electron Transport Complex II, Gene Expression, Glucuronidase, Glyceraldehyde-3-Phosphate Dehydrogenases, Humans, Integrin alpha6, Keratin-5, Keratinocytes, Lectins, Neoplasm Proteins, Phosphoglycerate Kinase, Psoriasis, Reference Values, Reverse Transcriptase Polymerase Chain Reaction, Ribosomal Proteins, S100 Calcium Binding Protein A7, S100 Proteins, Software, Toll-Like Receptors, Tumor Protein, Translationally-Controlled 1, beta 2-Microglobulin