8 protocols
AccessionType
 
array scanning protocol
mRNA profiling was carried out on Illumina HumanHT-12 Expression BeadChips
nucleic acid hybridization to array protocol
mRNA profiling was carried out on Illumina HumanHT-12 Expression BeadChips
nucleic acid labeling protocol
mRNA profiling was carried out on Illumina HumanHT-12 Expression BeadChips
treatment protocol
NHEK Keratinocytes were transfectred in 24-well tissue culture plates using 0.8 ul of siPORT NeoFX Transfection agent (Life technologies) and 50000 cells in 0.6 ml medium (Keratinocyte-SFM medium kit, Invitrogen). Keratinocytes were transfected for 24h and stimulated either with 10ng/ml IFN-gamma (R&D)for 48h. Transfections were performed at concentrations of 30 nM of pre-miRNAs (Pre-miR miRNA Precursors Molecules hsa-miR-146a and Negative Control #1, Life technologies)
nucleic acid extraction protocol
miRNAeasy Mini Kit (Qiagen) was used to purify RNA from primary keratinocytes. The concentration and quality of RNA was assessed with NanoDrop ND-1000 spectrophotometer and Agilent RNA 6000 Nano Kit on Agilent 2100 Bioanalyzer.
growth protocol
Pooled Normal Human Epidermal Keratinocytes (NHEK) from adult healthy donors (Promocell) were cultured in serum free KC medium (Keratinocyte-SFM; Invitrogen) supplemented with 5 ng/mL human recombinant epidermal growth factor, 50 mg/mL bovine pituitary extract in a humidified atmosphere containing 5% CO2 at 37 deg C. Medium was changed every second day, and cells between passages 2 and 5 were used in all experiments.
treatment protocol
NHEK Keratinocytes were transfectred in 24-well tissue culture plates using 0.8 ul of siPORT NeoFX Transfection agent (Life technologies) and 50000 cells in 0.6 ml medium (Keratinocyte-SFM medium kit, Invitrogen). Keratinocytes were transfected for 24h and stimulated with 25 ng/ml TNF-alpha (both from R&D)for 48h. Transfections were performed at concentrations of 30 nM of pre-miRNAs (Pre-miR miRNA Precursors Molecules hsa-miR-146a and Negative Control #1, Life technologies)