ABSTRACT: We used high density oligonucleotide arrays to measure the relative expression levels of ~27,000 probe sets in the left ventricles of inbred rat strains; DA (high performing), Copenhagen (COP, low performing), as well as F1(COPxDA) rats bred from these two strains whose performance was intermediate relative to both parental strains. DA and COP rats differ for both intrinsic aerobic running capacity (assessed with a treadmill exercise test to exhaustion) and intrinsic cardiac performance (as assessed by the Langendorff-Neeley working heart model). Previous work also showed a strong correlation between intrinsic aerobic running capacity and intrinsic cardiac performance. The inbred rats were maintained under specific pathogen free conditions and had access to pelleted rat chow (diet #5001, Purina Mills, Richmond, IN) and water ad libitim. The rats were housed with a target room temperature and humidity of 22oC and 40%, respectively, and 12:12 hour light dark cycle,with the light cycle occurring in the daytime (6 A.M. – 6 P.M.). The aerobic running capacity of the rats used in this study was assessed using a single week of treadmill endurance running tests, occurring at 11 weeks of age, and the rats were allowed 4 weeks to recover. Rats were killed at 15 weeks of age and their left ventricles were dissected. RNA was isolated by homogenizing the left ventricles of the heart in an ice cold guanidine thiocyanate/phenol solution (Ultraspec, Biotecx Laboratories, Houston, TX), extracted with chloroform and isolated using RNA Tack Resin (Biotecx Laboratories). Total RNA was further purified by ethanol precipitation and absorption to a column (RNeasy, Qiagen, Valencia, CA). Double-stranded cDNA was synthesized from 15 micrograms total RNA from each rat (four rats per strain) using reverse transcriptase (SuperScript II, InVitrogen, Carlsbad, CA) and T7-(dT)24 as the oligonucleotide primer. Biotinylated cRNA was synthesized using a kit (Enzo Bioarray High Yield RNA transcript Labeling Kit, Enzo Diagnostics, Farmingdale, NY) according to the manufacturer’s instruction and cRNAs were purified on columns (RNeasy mini kit, Qiagen). Each cRNA was fragmented according to the protocol in the Affymetrix GeneChip Expression Analysis manual and their quality was assessed by hybridization of a 5 microgram aliquot to a test chip (TestChip3, Affymetrix). 15 micrograms of the probe was hybridized to each of a set of three rat GeneChips (U34A, B, and C; Affymetrix). Hybridization, washing, staining with streptavidin-phycoerythrin, and scanning were performed at the Medical College of Ohio Core Facility according to the manufacturer's (Affymetrix) instructions. Complete transcription and hybridization were validated with bacterial sequences as an external control and several housekeeping genes as an internal control. Absolute analysis of data were conducted using the default settings of the Affymetrix software (GeneChip Software MAS-5.0). Microarray images were scaled to an average hybridization intensity of 150 to normalize the signals between individual chips. Keywords = quantitative real-time RT-PCR Keywords = insulin receptor substrate 2 (Irs2) Keywords = phosphatidylinositol 3-kinase Keywords = regulatory subunit 1 Keywords = (Pik3r1) Keywords = endurance running Keywords = treadmill running Keywords: parallel sample
