|
| Status |
Public on Nov 30, 2009 |
| Title |
Aortic valve Eln+/+ AV_WT_2 |
| Sample type |
RNA |
| |
|
| Source name |
Postnatal day 30 Eln+/+ aorta, postnatal day 30 Eln+/- aorta; postnatal day 30 Eln+/+ aortic valve; postnatal day 30 Eln+/- aortic valve
|
| Organism |
Mus musculus |
| Characteristics |
tissue: aortic valve
genotype: Elastin +/+
age: 30 days
|
| Treatment protocol |
None
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Heart tissue was dissected from 30 day old Eln+/+ and Eln+/- mice, and aortic valve and ascending aorta tissue was isolated and immediately submerged in Trizol [Invitrogen].
The quality of the Total RNA is checked by the Agilent Bioanalyzer 2100 (Hewelett Packard) using the RNA 6000 Pico Assay.
|
| Label |
biotin
|
| Label protocol |
1-20 ng of total RNA from each tissue specimen from each animal was used in an independent experiment (there was no tissue pooling). Use the WT- Ovation Pico RNA Amplification System Version 1.0 (Nugen) to prepare target cDNA. Then use the FL-Ovation cDNA Biotin Module V2 (Nugen) to fragment and biotin label the target cDNA.
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| |
|
| Hybridization protocol |
Full Protocol Description: Use a standard Probe Array Cartridge (GeneChip Mouse Genome 430 2.0 Array– Affymetrix). Create a hybridization cocktail for a single probe array that contains 5 mg of fragmented cRNA, 50 pM Control Oligonucleotide B2 (Affymetrix), 20X Eukaryotic Hybridization Controls (1.5 pM bioB, 5 pM bioC, 25 pM bioD, 100 pM cre) (Affymetrix), 10% DMSO, and 1X Hybridization Buffer. Heat hybridization cocktail to 99°C for 2 minutes, to 45°C for 5 minutes, and spin at maximum speed in a microcentrifuge for 5 minutes. Fill probe array with 200 mL of 1X Hybridization Buffer. Incubate at 45°C for 10 minutes in the GeneChip Hybridization Oven 640 (Affymetrix) rotating at 60 rpm. Remove 1X Hybridization Buffer and fill probe array with 200 mL of the hybridization cocktail. Incubate at 45°C for 18 hrs in the Hybridization Oven rotating at 60 rpm.
Fluidics: Wash and Stain probe arrays using the Fluidics Station 450 (Affymetrix) utilizing the fluidics protocol FS450-0004. Arrays were stained with phycoerythrin-conjugated streptavidin [Molecular Probes, Eugene, OR] and hybridization signals were amplified using antibody amplification with anti-streptavidin biotinylated antibody [Vector Laboratories, Burlingame, CA], as described in the Affymetrix GeneChip® Expression Analysis Manual.
|
| Scan protocol |
Images were scanned using a Genechip scanner 3000 [Affymetrix]
|
| Description |
Aortic valve tissue (consisting of cusp, annulus and root) and ascending aorta tissue was obtained from both Eln+/+ and Eln+/- mice (total 16 experiments).
|
| Data processing |
Affymetrix Gene Chip Mouse Genome 430 2.0 array gene chips were used. Gene Chip CEL files were subjected to RMA normalization using GeneSpring GX 7.3 software [Agilent]. Each gene was normalized to the median value of that gene.
Standard Affymetrix internal control genes were used to check the quality of the assay quality by the signals of the 3' probe set to the 5' probe set of the internal control genes, GAPDH and B-actin, with acceptable 3' to 5' ratios between1-3. Prokaryotic Spike controls were used to determine the hybridization of target RNA to the array occurred properly.
GeneSpring 7.3 (Agilent Technologies Inc. Palo Alto, California) was used for normalization and filtering. Data were analyzed using Ingenuity Pathway Analysis (Ingenuity® Systems, www.ingenuity.com). Fisher’s exact test was used to calculate a p-value determining the probability that the association between the genes in the dataset and the canonical pathway is explained by chance alone.
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| |
|
| Submission date |
May 07, 2009 |
| Last update date |
May 07, 2009 |
| Contact name |
Robert Bruce Hinton |
| E-mail(s) |
robert.hinton@cchmc.org
|
| Phone |
513-636 0902
|
| Fax |
513-636 5958
|
| Organization name |
Cincinnati Children's Hospital Medical Center
|
| Department |
Dept Of Pediatrics
|
| Street address |
3333 Burnet Ave
|
| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45236 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE16012 |
Gene expression profiles in Elastin (Eln+/-) mouse aorta and aortic valve tissue |
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