normalization data transformation protocol
GeneChip intensities were background-corrected, normalized and sumarized by RMA method, using the “Affy” package from Bioconductor. ID_REF = VALUE = Log2 RMA signal
array scanning protocol
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G
Following fragmentation, 10 µg of biotinylated cRNA were hybridized to Affymetrix chips. Each sample was added to a hybridization solution containing 100mM 2-(N-morpholino)ethanesulfonic acid, 1 M Na+, and 20mM of EDTA in the presence of 0.01% of Tween-20 to a final cRNA concentration of 0.05 µg/ml. Hybridization was performed for 16 h at 45ºC . Each microarray was washed and stained with streptavidin-phycoerythrin in a Fluidics station 450 (Affymetrix).
Biotinylated cRNA was prepared according to the standard Affymetrix protocol from 4 µg total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Bovine primary aortic endothelial cells were isolated from bovine aorta by collagenase treatment. Cells were grown on RPMI culture medium under standard culture conditions.
nucleic acid extraction protocol
Total RNA was extracted using RNeasy kit (QIAGEN).
sample treatment protocol
Primary aortic endothelial cells seeded on 10 mm diameter plates were incubated with TGF-beta 1 (5 ng/ml) for 24 hours or left under basal conditions.