|
| Status |
Public on Jan 01, 2011 |
| Title |
myocyte_serum containing media_adeno-bGal_sample2 |
| Sample type |
RNA |
| |
|
| Source name |
Primary culture of neonatal rat ventricular cardiomyocytes, serum containing media
|
| Organism |
Rattus norvegicus |
| Characteristics |
tissue: Neonatal cardiomyocytes
|
| Treatment protocol |
Neonatal cardiomyocytes were isolated from 1-3 day old Sprague Dawley rat pups using the Neonatal Cardiomyocyte Isolation System (Worthington) according to the manufacturer’s instructions. Briefly, hearts were rinsed with ice cold HBSS, and trypsinised for 16 hrs at 4oC. Hearts were incubated with trypsin inhibitor at 37°C, and then incubated for 60 minutes with a collagenase solution at 37°C with gentle mixing. Cell pellet was triturated and resuspended in M199 media (invitrogen) containing 10% fetal calf serum. Cells were then pre-plated for 1 hr to deplete culture of fibroblasts, and non adherent cells were plated on gelatin coated culture dishes. Following plating cells were washed twice with culture media containing 10% fetal calf serum, infected with adenovirus encoding for beta-Galactosidase and grown for 48 hrs.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using RNeasy Fibrous Tissue kit (Qiagen) according to the manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
The Affymetrix Whole Transcript Sense Target Labeling Assay (Affymetrix) is used to create biotin – labeled sense -strand cDNA targets for hybridization to the Gene 1.0 ST Arrays.
|
| |
|
| Hybridization protocol |
Hybridization cocktail for a single probe array contained 2.5 ?g 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), 7% DMSO, and 1X Hybridization Buffer. GeneChip Rat Gene 1.0 ST Array– Affymetrix was hybridized at 45?C for 18 hours in the GeneChip Hybridization Oven 640 (Affymetrix)
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G
|
| Description |
Gene expression data from cardiomyocytes in 10% serum containing media, infected with adeno-beta-galactosidase
|
| Data processing |
The data were analyzed with GeneSpring version 7.3 using RMA normalization, then normalized to median of all samples.
|
| |
|
| Submission date |
Jan 06, 2010 |
| Last update date |
Jan 01, 2011 |
| Contact name |
izhak kehat |
| E-mail(s) |
izhak.kehat@cchmc.org
|
| Organization name |
CCHMC
|
| Street address |
3333 burnet ave
|
| City |
cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
| |
|
| Platform ID |
GPL6247 |
| Series (1) |
| GSE19771 |
Neonat cardiomyocytes_hypertrophy_HDAC4 |
|
