Thoracic aorta dissected, cut into 2mm rings, washed 12 times in serum free EBM (Cambrex BioScience) and cultured for 13 days in serum free-EBM, changing medium every third day.
On day 13 , recombinant human angiopoietin-1 protein (R&D Systems #923-AN) added to the culture at a final concentration of 100ng/ml in 0.5ml EBM containing 5ug/ml poly-His antibody (R&D Systems #MAB050).
Extracted molecule
total RNA
Extraction protocol
20 hours after treatment with Ang-1, four to six individual rings were combined, snap frozen in liquid nitrogen, crushed, and extracted with Trizol reagent (Invitrogen) followed by RNAEasy Micro kit (Qiagen) purification with DNAse I treatment. Total RNA eluted into DEPC water.
Label
Phycoerythrin
Label protocol
Standard Affymetrix
Hybridization protocol
Standard Affymetrix protocol
Scan protocol
Standard Affymetrix protocol
Description
RNA labeling, array hybridization, scanning, and initial data transformation done at the Center for Expression Arrays at the Univesity of Washington.
