|
| Status |
Public on Mar 08, 2010 |
| Title |
caPI3K sham 1413 |
| Sample type |
RNA |
| |
|
| Source name |
Constitutively active (ca) PI3K mutant mice - sham (control) group heart tissue
|
| Organism |
Mus musculus |
| Characteristics |
Organism: Mus musculus
Strain: FVB/N
Source organ: heart
caPI3K-sham: Constitutively active (ca) phosphoinositide 3 kinase (PI3K) mutant, transgene expressed in mouse heart. Control group. Over-expression of PI3K is considered good for the heart. Normal function and histology at baseline. Molecular signature reflecting a heart that is protective against a pathological insult. Some gene changes reflecting stress of surgical procedure.
|
| Biomaterial provider |
Dr. Julie McMullen, Baker Heart Institute, Melbourne, Australia
|
| Treatment protocol |
The sham (control) operation was performed on mice at 4 months of age. After 8 weeks, cardiac function of the mice were measured by echo cardiograph and heart was removed. Total RNA was extracted following the removal.
|
| Growth protocol |
The mice were on a standard chow diet, and were kept in temperature controlled cages (21 - 23 degree celcius) and maintained a 12 hour light/dark cycle.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Heart was homogenized individually using motar and pestile in liquid nitrogen. Total RNA from the samples was collected by the addition of of TRIzol® Reagent (Invitrogen). Cells suspended in TRIzol® reagent were disrupted by repeated pipetting and then transferred into microfuge tubes. This protocol was modified from manufacturer’s instructions and is described here. The lysate was further homogenized using syringe and needle for 30 s and incubated at room temperature (~21ºC) for 5 min, and 200 l of chloroform was added. The tubes were shaken vigorously for 15 s to mix the phases and incubated for 3 min at room temperature. The phases were separated by centrifugation at 12,000 x g for 15 min at 4ºC. The upper aqueous phase was transferred into a fresh microfuge tube and 500 µl of ice cold isopropanol added to precipitate the
RNA. The contents were mixed by gentle inversion and then incubated for 10 min at room temperature. The RNA was recovered by centrifugation at 12,000 x g for 10 min at 4ºC. A gelatinous pellet was left on the side of the tube after removal of the supernatant. The RNA pellet was washed with 1 ml of 75% ethanol and the tube was vortexed briefly to resuspend the pellet. The RNA was recovered by centrifugation at 7,500 x g for 5 min at 4ºC. This washing step was repeated twice. After removal of the supernatant, the RNA pellet was airdried briefly and then resuspended in 20 µl Nuclease-free H2O (Ambion Inc., Austin, TX, USA) and stored in -80ºC for further processing.
|
| Label |
biotin
|
| Label protocol |
cRNA targets for the Affymetrix arrays were synthesized, labeled, and purified according to vendor's (Affymetrix) instructions.
Please refer to Expression Analysis Technical Manual available from Affymetrix website
(GeneChip® Expression Manual, Affymetrix, Inc., Santa Clara, CA)
|
| |
|
| Hybridization protocol |
The Affymetrix arrays were hybridized and washed using the manufacturer's protocol. The arrays were then stained with
streptavidin-phycoerythrin using the standard antibody amplification protocol.
Please refer to Expression Analysis Technical Manual available from Affymetrix website
(GeneChip® Expression Manual, Affymetrix, Inc., Santa Clara, CA)
|
| Scan protocol |
Affymetrix arrays were scanned with the Affymetrix GeneArray scanner.
Please refer to Expression Analysis Technical Manual available from Affymetrix website
(GeneChip® Expression Manual, Affymetrix, Inc., Santa Clara, CA)
|
| Description |
Constitutively active PI3K transgenic mice treated with sham operation to serve as controls. Array - 1413.
|
| Data processing |
The images were analyzed using Microarray Suite 5.0 software (MAS5.0; Affymetrix Inc., Santa Clara, CA). Expression ratios were computed by comparative analysis of MAS5 values. The data were filtered using MAS5's signal detection and change calls generated at recommended default settings. The ratios included were those that had present calls for signal detection or an increase or decrease call associated with the ratio
|
| |
|
| Submission date |
Apr 10, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Ruby CY Lin |
| E-mail(s) |
rubyl@unsw.edu.au, ruby.lin@sydney.edu.au
|
| Organization name |
The Westmead Institute for Medical Research
|
| Street address |
176 Hawkesbury Road, Westmead
|
| City |
Sydney |
| State/province |
NSW |
| ZIP/Postal code |
2145 |
| Country |
Australia |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE7487 |
Gene profiling of pathological cardiac hypertrophy vs physiological hypertrophy |
|
| Relations |
| Reanalyzed by |
GSE119085 |
