|
| Status |
Public on Oct 13, 2012 |
| Title |
Heart_KO_2 |
| Sample type |
RNA |
| |
|
| Source name |
heart tissue, ad lib feeding, 10-12 weeks old
|
| Organism |
Mus musculus |
| Characteristics |
gender: male
strain: C57B/6Jx129Sv
genotype/variation: SRC-2 knockout (KO)
tissue: heart
age: 10-12 weeks old
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen hearts isolated from WT and SRC-2 KO animals were homogenized in buffer RLT and subsequent RNA was isolated using the RNEasy kit according to the manufacturer’s protocol (Qiagen).
|
| Label |
biotin
|
| Label protocol |
250ng of RNA isolated from total heart (RNeasy kit, Qiagen) for each sample was labeled using the new standard Affymetrix linear amplification protocol using the 3' IVT Express Kit. This was reverse-transcribed and cRNA was produced and biotinylated via in vitro transcription.
|
| |
|
| Hybridization protocol |
A hybridization cocktail containing Affymetrix spike-in controls and 15 μg fragmented, labeled cRNA was loaded onto a GeneChip® Mouse 430 2.0 array. The array was hybridized for 16 hours at 45°C with rotation at 60 rpm then washed and stained with a strepavidin, R-phycoerythrin conjugate stain using the FS 450_0001 Fluidics protocol setting.
|
| Scan protocol |
Signal amplification was done using biotinylated antistreptavidin. The stained array was scanned on the Affymetrix GeneChip® Scanner 3000. The images were analyzed and quality control metrics recorded using Affymetrix Command Console v3.
|
| Data processing |
We used the following software packages for data QC, statistical analysis and presentation of the results: Affymetrix Expression Console (www.affymetrix.com), Partek (www.partek.com), BRB Array Tools (linus.nci.nih.gov/BRB-ArrayTools.html), and dChip (biosun1.harvard.edu/complab/dchip). Expressions were estimated using the RMA (Multi-Array Analysis) method [38] with Partek software.
Differentially expressed genes were found using the RVM (Random Variance Model) t-test, which is designed for small sample size experiments [39]. We used BRB Array Tools software, developed by Dr. Richard Simon and the BRB-ArrayTools Development Team. All genes were included in the comparison. For the genes represented by more than one probeset, we used the most highly expressed probeset. The cutoffs for differentially expressed genes were False Discovery Rate (FDR) = 0.05.
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| |
|
| Submission date |
Oct 12, 2012 |
| Last update date |
Oct 13, 2012 |
| Contact name |
Erin Reineke |
| Organization name |
Baylor College of Medicine
|
| Street address |
One Baylor PLaza
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE41558 |
SRC-2 Coactivator Deficiency Decreases Functional Reserve in Response to Pressure Overload of Mouse Heart |
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