|
| Status |
Public on Aug 26, 2011 |
| Title |
AVIC sample#3 treated with DAPT |
| Sample type |
RNA |
| |
|
| Source name |
aortic valve cells treated with DAPT
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Sprague-Dawley
cell type: aortic valve interstitial cells
|
| Treatment protocol |
Not applicable.
|
| Growth protocol |
Rat aortic valve cusps were harvested from adult Sprague-Dawley rats and AVICs were cultured from explants based on published protocols. Briefly, valve leaflets were subjected to collagenase digestion and gently scraped to expose the subendothelial layer. The leaflets were then cut into microscopic pieces (1-2 mm2) and cultured in standard Medium-199 supplemented with 15% FBS, 2 mmol/L glutamine and 100 U/ml penicillin/streptomycin. Upon reaching 80% confluency, AVICs were passaged using trypsin-EDTA. AVICs between passage 3 and 8 were used for experiments. Notch signaling was inhibited using a γ-secretase inhibitor (Sigma Cat. # S2188 or D5942) diluted in DMSO to a concentration of 10 μM. Culture media was changed every 48-72 hours and cells were passaged at each time point of harvest.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Following fragmentation, labeled cRNA was hybridized to the Rat Genome 230 2.0 Array. Hybridization was allowed to continue for 16 hours at 45ºC followed by washing and staining of microarrays in a Fluidics Station 450 (Affymetrix Inc., USA).
|
| |
|
| Hybridization protocol |
GeneChips were scanned in a GeneChip Scanner 3000 (Affymetrix Inc., USA)
|
| Scan protocol |
CEL files were generated from DAT files using GeneChip® Operating Software (GCOS) software (Affymetrix Inc., USA). The probe set signals were generated using the RMA algorithm in ArrayAssist 3.4 (Stratagene) and were used to determine differential gene expression by pair-wise comparisons. The genes that were altered by two-fold either ways were sorted and used for further interpretation of the microarray data.
|
| Description |
Gene expression data from DAPT treated mouse replicate 3
|
| Data processing |
Data were RMA normalized and pairwise comparisons (of averaged signal values) and Student’s t test with Benjamin and Hoschberg adjustment were performed using GeneSifter (VizX Labs, Seattle, WA). Genes with an average fold-change ≥ 1.5 and an adjusted p value ≤ 0.05 were considered significantly differentially expressed.
|
| |
|
| Submission date |
Aug 25, 2011 |
| Last update date |
Aug 26, 2011 |
| Contact name |
Vidu Garg |
| E-mail(s) |
[email protected]
|
| Phone |
614-355-5710
|
| Organization name |
Research Institute at Nationwide Children's Hospital
|
| Department |
Cardiovascular Research
|
| Lab |
Vidu Garg
|
| Street address |
700 Children's Drive WB4239
|
| City |
Columbus |
| State/province |
OH |
| ZIP/Postal code |
43205 |
| Country |
USA |
| |
|
| Platform ID |
GPL1355 |
| Series (1) |
| GSE31668 |
Inhibitory Role of Notch1 in Calcific Aortic Valve Disease |
|
