Hearts from 1-2 days old Wistar rats (Charles River, Sulzfeld, Germany) were harvested and minced in PBS. Subsequently, up to six digestion steps were carried out with pancreatin (Sigma, Munich, Germany, 0,6 mg/ml) and collagenase type II (Worthington, 0,5 mg/ml) in sterile ADS buffer containing 120 mmol/l NaCl, 20 mmol/l HEPES, 8 mmol/l NaH2PO4, 6 mmol/l glucose, 5 mmol/l KCl, 0.8 mmol/l MgSO4, pH 7.4. Cardiomyocytes were purified from contaminating fibroblasts using a Percoll (Amersham, Germany) gradient centrifugation step. Finally, NRVCMs were resuspended and cultered in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS, penicillin/streptomycin and L-glutamine (all from PAA, Linz, Austria) .
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from NRVCMs using the TRIzolTM reagent (Invitrogen, Germany) according to the manufacturer’s protocol. RNA was resuspended in DEPC (Sigma)–treated H2O. RNA integrity and purity were electrophoretically verified by ethidium bromide staining and measurement of OD260/OD280 absorption ratios. Total RNA was pooled from several preparations and purified from contaminating genomic DNA by a DNase I digestion (Sigma, AMPD1).
Label
biotin + phycoerythrin-streptavidin
Label protocol
Affymetrix expression profiling was carried out at the German Ressource Center for Genome Research (RZPD; Berlin, Germany). Briefly, integrity of RNA was checked using an Agilent Bioanalyzer (Eukaryote total RNA assay) followed by cDNA synthesis and in vitro transcription with Ambion’s Message Amp kit according the manufacturer’s instructions. cRNA was subjected to quality control using the Bioanalyzer with the mRNA smear nano assay. Final cRNA concentration was 60 ng/µl.
Hybridization protocol
Hybridization was carried out for 16-18 hours in the Affymetrix Oven 640 at 60 rpm and 45°C. Subsequent washing and staining was performed with a fluidics station (Gene Chip® Fluidics station 400, Affymetrix) and controlled by Affymetrix GeneChip Operating System (GCOS).
Scan protocol
The scanning process and primary data analysis was done with the Affymetrix GeneChip Scanner 3000 controlled with GCOS.
Description
control condition 1
Data processing
variance stabilizing normalization procedure (Huber et al., Bioinformatics 2002)
