Primary cardiomyocyte cultures were obtained from the ventricles of newborn Wistar rats by the method of Yagev et al.: some cells were grown in HAM F10 plus 10% fetal calf serum and 10% horse serum medium (controls), others in the same medium supplemented with 60 M Eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). Media were changed every 48 hrs; cells were grown until complete confluence, then they were scraped off in ice cold PBS, and RNA isolation, labeling of complementary RNA (cRNA), hybridization to 22K-gene arrays (Rat oligo array G4130A) and assessment of expression ratios were performed. About one million cells treated with RNAlater were homogenized and total RNA was extracted by column technology (Rneasy Protect mini kit) and analyzed on both a spectrophotometer and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Only samples with 28S/18S ratio of >2.0 and no evidence of ribosomal peak degradation were included. The cRNA was generated by in vitro transcription with the use of T7 RNA polymerase (Low RNA input fluorescent linear amplification kit) and labeled with Cy3-CTP or Cy5-CTP.
