6 protocols
Title: Affymetrix CEL analysis. Description:
hybridization protocol
Affymetrix Generic Hybridization
nucleic acid labeling protocol
Labeling of samples was carried out according to the manufacturer’s standard protocol
Total RNA was extracted using the RNeasy kit as specified by the manufacturer (Qiagen Ltd, Crawley, UK). RNA concentration was measured using ND-1000 Nanodrop (Thermo Scientific, Wilmington, USA). The quality was assessed by running the samples on the RNA 6000 LabChip kit (Agilent Technologies, Waldbronn, Germany) with the Agilent 2100 bioanalyzer. 500 ng of total RNA was amplified using the Ambion WT Expression Kit (Affymetrix).
growth protocol
Primary mouse vascular smooth muscle cells were obtained by enzyme digestion of excised aortae from 5-w-old wild-type C57BL/6 mice. Cells were seeded at a density of 1.0x104 cells/cm2 in growth medium consisting of alpha-MEM (Invitrogen, Paisley, UK) supplemented with 10% FCS (Invitrogen) and 1% gentamycin (Invitrogen). Cells were cultured in a humidified atmosphere of 95% air/5% CO2 at 37 degrees celsius.
treatment protocol
At confluency (day 0), growth medium was supplemented with 2.5 mM B-glycerophosphate (BGP) and 50ug/ml ascorbic acid to induce matrix calcification. Cells were sampled at day 0 and 9