title: Meaning of relative gene expression in multilayered cultures of epidermal keratinocytes.
abstract: Reconstructed human epidermis (RHE) has become an in vitro model of choice for studying cell and tissue functions. Analysis of gene expression over the course of reconstruction must take into account the heterogeneous differentiation states of keratinocytes reconstituting the typical epidermal layers. In monolayer cultures, relative mRNA expression levels of differentiation markers are usually expressed as a ratio versus a classical reference gene (also named house-keeping gene) tested to be expressed equally in certain experimental conditions. Applied to complex tissues in which the cell number increases over time together with differentiation, calculation of relative gene expression does not take enough into account a crucial phenomenon: epidermal morphogenesis results in progressive restriction of differentiation markers, such as involucrin, to a specific layer, or in the delayed onset of mRNA expression of filaggrin or TMEM45A for instance following stratification. Our study illustrates that comparing the relative expression level of mRNAs to that of a basal layer-specific gene (e.g. ITGA6) better illustrates the contribution of specific differentiation markers to the process of epidermal morphogenesis.
authors.affiliation: Cell and Tissue Laboratory, URPHYM-NARILIS, University of Namur, Namur, Belgium.
authors.forename: Jérémy
authors.initials: J
authors.lastname: Malaisse
authors.forename: Maryse
authors.initials: M
authors.lastname: Hermant
authors.forename: Aurélie
authors.initials: A
authors.lastname: Hayez
authors.forename: Yves
authors.initials: Y
authors.lastname: Poumay
authors.forename: Catherine
authors.initials: C
authors.lastname: Lambert de Rouvroit
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D000078404:Epidermal Cells; D004817:Epidermis / Q000378:metabolism*; D000091344:Filaggrin Proteins; D015870:Gene Expression; D006801:Humans; D039503:Integrin alpha6 / Q000235:genetics; D007381:Intermediate Filament Proteins / Q000235:genetics; D053550:Keratin-10 / Q000235:genetics; D053547:Keratin-14 / Q000235:genetics; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics
chemical_list: C000717249:FLG protein, human; D000091344:Filaggrin Proteins; C588002:ITGA6 protein, human; D039503:Integrin alpha6; D007381:Intermediate Filament Proteins; C507493:KRT10 protein, human; C507540:KRT14 protein, human; D053547:Keratin-14; D012333:RNA, Messenger; C402487:RPL13A protein, human; D012269:Ribosomal Proteins; D053550:Keratin-10
keywords: 3D-cultures; Keratinocytes; RT-qPCR; reconstructed human epidermis; relative expression