Molecular Mapping of Keratinocyte Differentiation by Functional Gene Expression Profiling. Homo sapiens

Our goal was to establish a robust and reliable platform for the mapping of gene expression changes occurring during the process of keratinocyte differentiation. We hypothesized that a functional gene expression profiling approach capable of identifying the program of gene expression over time would facilitate more focused identification of relevant genes. To achieve this, we used the confluenceinduced differentiation (CID) model, a widely used and reliable method that in many aspects recapitulates in vivo differentiation of human epidermal keratinocytes (Paragh et al, 2010; Poumay and Pittelkow, 1995). Overall design: Primary human keratinocytes from three donors were subjected to CID in separate cultures and harvested at successive time points including 2 days prior to reaching confluence (D(-2)), at the point of confluence (D(0)), as well as after reaching confluence (D(1), D(2) and D(3)). In order to minimize any donor and sample effects, the RNA samples were pooled at each time point and gene expression profiling was performed (Affymetrix U133 A & B). Secondary validation study was performed with three additional time observation arrayed in Affymetrix U133A2 platform for the 3 patient pool. The time observations were: D(-3),D(-2),D(-1),D(0),D(+1),D(+2),D(+3),D(+5).