Meaning of relative gene expression in multilayered cultures of epidermal keratinocytes

Malaisse, Jérémy; Hermant, Maryse; Hayez, Aurélie; Poumay, Yves; Rouvroit, Catherine Lambert de

doi:10.1111/exd.12501

Cited by 6 publications

(5 citation statements)

References 11 publications

(19 reference statements)

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“…Recently, ITGA6 has been identified as a differentiation depended RG in organotypic cell cultures that is selectively expressed in the basal layer of the epidermis . Our results are in line with those of Malaisse and co‐workers, showing that the relative expression of ITGA6 decreases during differentiation.…”

Section: Discussionsupporting

confidence: 91%

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

Beer

Mlitz

Gschwandtner

et al. 2015

Experimental Dermatology

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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.

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Section: Discussionsupporting

confidence: 91%

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

Beer

Mlitz

Gschwandtner

et al. 2015

Experimental Dermatology

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“…With respect to the bound lipid profile, this is similar to that of native human skin (Ponec et al, 2003) however, it must be stated that measurements of this profile have been conducted with a lower level of precision. Furthermore, the presence of a leaky cornified envelope would also contribute to a reduced skin barrier property; while almost no data have been reported to date on the protein composition of the cornified envelope in HSEs, it has been shown that some cornified envelope proteins are expressed differently (involucrin) in the models as compared to native human skin (Malaisse et al, 2014).…”

Section: Tab 1: Models For Dermal Absorptionmentioning

confidence: 99%

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

Gordon

Daneshian

Bouwstra

et al. 2015

ALTEX

122

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SummaryModels of the outer epithelia of the human body -namely the skin, the intestine and the lung -have found valid applications in both research and industrial settings as attractive alternatives to animal testing. A variety of approaches to model these barriers are currently employed in such fields, ranging from the utilization of ex vivo tissue to reconstructed in vitro models, and further to chip-based technologies, synthetic membrane systems and, of increasing current interest, in silico modeling approaches. An international group of experts in the field of epithelial barriers was convened from academia, industry and regulatory bodies to present both the current state of the art of non-animal models of the skin, intestinal and pulmonary barriers in their various fields of application, and to discuss research-based, industry-driven and regulatory-relevant future directions for both the development of new models and the refinement of existing test methods. Issues of model relevance and preference, validation and standardization, acceptance, and the need for simplicity versus complexity were focal themes of the discussions. The outcomes of workshop presentations and discussions, in relation to both current status and future directions in the utilization and development of epithelial barrier models, are presented by the attending experts in the current report.

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“…Epidermal proteins analysed in this work have been selected from tissue microarray data available in the Human Protein Atlas database (http://www.proteinatlas.org) or from the literature. More precisely, the following proteins were described in some studies as specifically expressed in a single epidermal layer: LFNG, ITA6, KLK14, KLK5, KLK7, PRSS8, PAR2, RFNG [17][18][19][20][21], CADH3 [22], TGM1 [23], K2C5, K1C14, K1C10, H6VRG3, TGM2, INVO, LORI, SPRR4, CRNN, FILA [24], E9PBK1 [25], VDR, CP27B [26]. Liver proteins expressed preferentially in periportal or perivenous epatocytes were selected based on previous works [15,16,27 -29].…”

Section: Identification Of Protein Markersmentioning

confidence: 99%

The analysis of glutamate and glutamine frequencies in human proteins as marker of tissue oxygenation

et al. 2019

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In this study, we investigated whether the relative abundance of glutamate and glutamine in human proteins reflects the availability of these amino acids (AAs) dictated by the cellular context. In particular, because hypoxia increases the conversion of glutamate to glutamine, we hypothesized that the ratio glutamate/glutamine could be related to tissue oxygenation. By histological, biochemical and genetic evaluation, we identified proteins expressed selectively by distinct cellular populations that are exposed in the same tissue to high or low oxygenation, or proteins codified by different chromosomal loci. Our biochemical assessment was implemented by software tools that calculated the absolute and the relative frequencies of all AAs contained in the proteins. Moreover, an agglomerative hierarchical cluster analysis was performed. In the skin model that has a strictly local metabolism, we demonstrated that the ratio glutamate/glutamine of the selected proteins was directly proportional to oxygenation. Accordingly, the proteins codified by the epidermal differentiation complex in the region 1q21.3 and by the lipase clustering region 10q23.31 showed a significantly lower ratio glutamate/glutamine compared with the nearby regions of the same chromosome. Overall, our results demonstrate that the estimation of glutamate/glutamine ratio can give information on tissue oxygenation and could be exploited as marker of hypoxia, a condition common to several pathologies.

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Meaning of relative gene expression in multilayered cultures of epidermal keratinocytes

Cited by 6 publications

References 11 publications

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

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

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

The analysis of glutamate and glutamine frequencies in human proteins as marker of tissue oxygenation

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