4 protocols
Cells were serum starved (0% serum) for 4 hours then treated with 10ng/ml IFN-alpha2b (Promokine, Germany) or vehicle control in EGM-MV2 5%FCS for 6 hours.
Human Arotic Endothelial Cells (HAoECs) from a single donor (Promocell, Germany) were cultured in EGM-MV2 5% FCS in uncoated plastic ware in standard culture conditions (5% CO2, humidified environment). Cells used at passages 6 and 7. (Parameters: time unit = seconds, min temperature = 37, temperature unit = C, media = EGM-MV2)
Exons of the Core Meta-Probeset were used. Quantile Normalisation and RMA background correction was performed. Data analysis performed with Partek Genomics Suite, version 6.3, Copyright 2007, Partek Inc., St. Charles, MO, USA.
Cells were lysed in 1ml TRI Reagent (Sigma-Aldrich) followed by RNA extraction according to the manufacturer’s instructions. To remove any genomic DNA, RNA was treated with DNase I (Ambion) at 37C for 30 mins and the reaction stopped by addition of Phenol:Chloroform:IAA. Following centrifugation at 12,000xg for 5 mins at 4C, the aqueous phase was removed and RNA precipitated during a 30 minute incubation on ice with 1/10 ammonium acetate (Ambion) and 3x 100% ethanol. Centrifugation at 12,000xg for 20 mins at 4C produced an RNA pellet which was washed in 75% ethanol, centrifuged again for 5 minutes, before air-drying and dissolving in tris-EDTA buffer (Ambion). (Parameters: Extracted product = total_RNA, Amplification = none)