In vivo study, aortic valve, pulmonary artery, aorta and spleen were isolated from mice in 3 groups: 1) intraperitoneal injection of 20mg of LPS priming only, 2) oral administration of FK565 (100mg) for consecutive days 3) oral administration of FK565 (100mg) for consecutive days 1 day after LPS priming, at day 2, 4, and 7. In vitro study, each tissue of aortic valve, pulmonary artery, aortic arch, and abdominal aorta (between roots of right and left renal arteries) sterilely isolated from mice (BALB/c) was cultured in a 96-well plate with EBM-2 medium and EGM-2 (Lonza) in the presence of FK565 (10μg/mL, N=6) or none (N=6) in a CO2 (5%) incubator at 37°C for 24 hours.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the RNeasy Fibrous Tissue (Qiagen Inc., Valencia, CA, USA) and amplified using Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion, Austin, TX, USA). RNA quality and concentration was determined by analysis with NanoDrop at the Harvard-Partners Center for Genetics and Genomics (Harvard Medical School, Cambridge, MA USA).
Label
Cy3
Label protocol
Nimblegen one-color cy3 labeling
Hybridization protocol
Hybridization was performed for 19h at 42C to the mouse Nimblegen Gene Expression arrays (090901_MM9_EXP_HX12, Roche NimbleGen, Madison, WI, USA) .
Scan protocol
GeneChips were scanned using the Gene Pix 4000B (Molecular Devices Corporation) .
Description
This sample is of pulmonary artery of mice(BALB/c) which was oral administrated of FK565 (100ug) for consecutive days. Aortic valve were collected at day 2.
Data processing
The raw data (tif.) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan software package, version 2.5. (Roche NimbleGen, Inc.).
