Evaluation of Keratinocyte Gene Expression

A Roche LightCycler480 instrument with 384-well microplates was used for this evaluation test of gene expression involved in keratinocytes epidermal physiology. Normal Human Epidermal Keratinocytes were grown and amplified to produce cells for the evaluation. Forty-eight-well microplates were seeded with cells (50,000 cells per well) and incubated for 48 h in a temperature-, humidity-, and CO₂-controlled environment. Samples were added on the cells while renewing the culture medium and were further incubated for 24 h. After incubation, the cells were washed and frozen at −80 °C to preserve the RNA. The RNAs were then extracted and quantified, and their quality was checked before performing their reverse transcription into cDNA. An RT-qPCR was finally performed for each experimental condition for the quantification of the expression of a set of 16 selected genes related to the barrier function (Claudin 1, Cornifelin, Desmoglein 1, Kallikrein-related peptidase 7, Tight junction protein 1), epidermal renewal (Epiregulin, Hyaluronic acid synthase 3, Heparin-binding EGF-like growth factor, Keratin 19), keratinocyte differentiation (Keratin 10, Small prolin-rich protein A1, Transglutaminase 1), and stress response (Glutathione peroxidase 2, Heme oxygenase 1) in keratinocytes. The gene expression was measured on the highest non-toxic dose of each tested sample with a maximal dose of 0.2 g/L or 0.2 mM. The maximum non-cytotoxic dose was determined prior to the gene-expression testing at a dose of 0.2 g/L using a biological model under the same incubation conditions. All samples were evaluated at the same concentration in addition to a five-times-lower dose in a one-step protocol. The fold changes (FC) were calculated after a double normalization against the housekeeping genes and non-treated condition. Fold changes of gene expression were considered as modulated over 1.5 (induction) or under 0.5 (repression).