Differential and overlapping effects of 20.23(OH)2D3 and 1,25(OH)2D3 on gene expression in human epidermal keratinocytes. Differential and overlapping effects of 20.23(OH)2D3 and 1,25(OH)2D3 on gene expression in human epidermal keratinocytes

Neonatal keratinocytes from African American donors of passage 2 or 3 were treated with 20,23(OH)2D3, 1,25(OH)2D3 or 0.1% ethanol (control) for 6 and 24 hours. The cells were harvested separately, RNA isolated and submitted for microarray analysis at the Molecular Resources Center at the UTHSC. Overall design: Neonatal foreskins of African American donors were used to isolate neonatal human epidermal keratinocytes. Petrie dishes (100 mm in diameter) were seeded with human neonatal keratinocytes that were combined from 4 different black donors of either passage 2 or 3. After reaching 70-80% of confluence cells were treated with 10-7M of either 20,23(OH)2D3 or 1,25(OH)2D3 or 0.1% of ethanol (EtOH solvent control) for 6 or 24 hours. After 6 or 24 h of incubation cells were isolated from 3 plates per each experimental condition and combined for passage 2 and 3, separately. The RNA from HEK treated with either 20,23(OH)2D3 or 1,25(OH)2D3 or 0.1% ethanol (control), was isolated using the Absolutely RNA Miniprep Kit (Stratagene). High purity RNA samples were submitted for microarray analysis at the Molecular Resources Center at the UTHSC. Expression profiling was accomplished using whole-genome gene expression direct hybridization assay using Illumina’s HumanWG-6_V2 (Platform GPL13376) chip/array (Illumina, San Diego, CA).