Ambrosino series 1

Hybridizations were performed in triplicate, using total of 7 RNA samples extracted from 3 independent batches of proliferating cardiomyocyte cell line derived from p38alpha knockout mouse. In two hybridizations (Ambrosino 1, Ambrosino 2), the RNAs from p38alpha-/- and wt cells were labeled with Cy-5 and Cy-3-dUTP, respectively, whereas in the third hybridization (Ambrosino 3), the dyes were swapped. The multiple spike-in control RNA templates were added to the sample upon direct labeling, and the successful labeling and hybridization was confirmed in each hybridizations. Twelve housekeeping genes in the array (GAPDH, HPRT and S16, S9, and S8 ribosomal proteins) gave average value of Ratio of Medians (ROM), 1.30 ± 0.26, which was confirmed to be acceptable. GenePix Pro program calculates the Normalization Factor, based on the premise that the arithmetic mean of the ratios from every feature on the given array should be equal to 1. Normalization was therefore performed by multiplying the Factor to ROM in each gene. The program also identifies features that did not give good alignment to the expected spotted area as “flagged” spots, indicative of the impaired-quality hybridization of the specific genes. All flagged genes were removed from the list before further analysis. The genes that passed all these criteria were sorted by ROM and those that showed more than ±1.5 fold changes were selected in the data table from each hybridization. Finally, the data tables from the 3 independent hybridizations were compared and only those genes that appeared in all 3 experiments were selected in the final list (manuscript submitted). Keywords = NIA mouse 15K Keywords = cardiomyocytes Keywords = p38alpha MAPK Keywords: repeat sample