Proteins extracted from myocardium with AMI (MI proteins) were added to culture media with concentration of 25 ug/ml. Additionally, oscillating pressure was applied with specially designed oscillating pressure chamber to simulate intra-cardiac pressure change. Cells were harvested after three days of exposure to MI protein and one day in oscillating pressure chamber.
Growth protocol
mononuclear cells were isolated by centrifugation (400G, 50 min) in a Ficoll hypaque gradient (density 1.077 g/cm3, Sigma). And these cells were cultured in α-minimum essential medium (α-MEM, Gibco BRL, Carlsbad), supplemented with 10% FBS (Gibco BRL, Carlsbad), 10,000 units/ml penicillin G sodium, 10,000 ug/ml streptomycin sulfate, and 25 ug/ml amphotericin B (Invitrogen Co.) at a concentration 5.0 x 106 cells/cm2. Cultures were maintained in a humidified 37℃ cell incubator with 5% CO2 with a change of culture medium twice a week. Three weeks later, monolayer of fibroblast-like adherent cells were trypsinized (0.25% trypsin, HyClone), washed, resuspended in culture medium and subcultured at a concentration of 1.0 x 106 cells/cm2
Extracted molecule
total RNA
Extraction protocol
Total RNAs from UCB-MSCs were isolated using TRIzol (Invitrogen, Carlsbad) and the RNeasy Mini kit (Qiagen, Valencia) as described previously
Label
biotin
Label protocol
cDNA was synthesized from total RNA using the One-Cycle cDNA Synthesis Kit. After clean up with a Sample Cleanup Module, double strand cDNA was used for in vitro transcription (IVT). cDNA was transcribed using the GeneChip IVT Labeling Kit, in the presence of biotin-labeled CTP and UTP. After clean up with a Sample Cleanup Module, labeled cRNA was fragmented by fragmentation buffer.
Hybridization protocol
Fragmented cRNA was hybridized to the Affymetrix Human Genome U133 Plus 2.0 Array.
Scan protocol
After hybridization, the arrays were washed in a GeneChip Fluidics Station 450 with wash buffer. After washing, the arrays were stained with a streptavidin-phycoerythrin complex. After staining, intensities were determined with a GeneChip scanner 3000, controlled by GCOS Affymetrix software.
Description
Gene expression data from UCB-MSC M02 naïve state
Data processing
As a normalization process, RMA (Robust Multi-Array Average) normalization was applied to remove systematic variance. By applying RMA, the raw intensity values are background corrected, log2 transformed and then quantile normalized.