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

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.

Chronic sun exposure is associated with distinct histone acetylation changes in human skin

“…Beer et al . found that GUSB mRNA was the most stably expressed gene in human primary keratinocytes in response to UV irradiation, and therefore we chose GUSB as an endogenous control for normalization of target gene expression. The primers are listed in Table S1 (see Supporting Information).…”

Epidermal reference genes at the forefront of data interpretation

“…In this issue of Experimental Dermatology, Beer et al. have exhaustively compared and combined different methods recommended for choosing adequate references used in gene expression analysis. This new study is carefully and thoroughly performed.…”

“…Interestingly, this new report further reveals for the first time that glucuronidase beta is a highly reliable reference gene in keratinocytes, drawing additional attention on glycosaminoglycan catabolism and on the stability of related degradation enzymes during keratinocyte differentiation. Finally, a very useful summary of recommended reference genes is provided, which should provide valuable context to future studies in this field .…”

“…Each author has first read the paper by Beer et al. and then all of them shared their individual comments to collectively construct this commentary, which was finally compiled by YP. All authors have read and approved its final version.…”

Genome Based Search for Reference Genes for Gene Expression Analysis in Oral Cancer: a Data Science Driven Approach

“…Most of the initial work [17] focussed on evaluating a set of known reference gene candidates for stability of expression using several normalization algorithms -geNorm [18], NormFinder [19] and bestKeeper [20]. However, researchers also tried assessing gene stability using bioinformatics approach [21], statistical measures like the CV [22], and the difference in DNA entropies in promoters driving the expression of specific genes [23].…”