weighed and then frozen in liquid nitrogen and stored at 280uC. Preparation of cRNA, direct labeling and oligonucleotide array hybridization Total RNA, isolated from LETO and OLETF hearts transduced with Ad.SERCA2a or Ad.b-Gal, was amplified and the cyanine-3/cyanine-5 labeled CTP was incorporated using T7 RNA polymerase. Equimolar 
amounts of cRNAs from control and DM, DM+SERCA2a or DM+b-Gal hearts were mixed together and were cleaned through QIAquick PCR Purification Kit spin columns to remove unincorporated dye-labeled nucleotides. The concentration and pmol incorporation was calculated after absorbance readings were taken at 260, 280, 550 and 650 nm. To perform reverse labeling, equimolar amounts of oppositely labeled cRNAs from the infected and uninfected hearts were mixed and hybridized to a separate microarray slide. The slides were hybridized, washed and scanned according to Agilent recommendations and settings. Scanning and Feature Extraction of arrays Arrays 7510950 were scanned with Agilent’s G2565AA Microarray Scanner System and analyzed using Agilent Feature extraction software, which calculates log ratios and P values for valid features on each array and provides a confidence measure of gene differential expression by performing outlier removal, background subtraction, and dye normalization for each feature. The software filters features that are not positive and significant with respect to background or features that are saturated. It then fits a normalization curve across the array using the locally weighted linear regression curve fit algorithm to detect and correct dye bias. Materials and Methods Construction of recombinant adenoviruses For the generation of E1 deleted SERCA2a and b-galactosidase adenoviruses we used the pAdEasy-1 adenoviral plasmid and the pAdTRACK shuttle vector, containing green fluorescent protein under the control of the CMV promoter. The Adenovirus b- galactosidase was used 20032260 as a control. The titers of stocks used in these studies measured by plaque assays were 5.961010 pfu/ml and 4.561010 pfu/ml, with a particle/pfu ratio of 40:1. Wild-type adenovirus contamination was excluded by the absence of PCR-detectable early region 1 sequences. Analysis of microarray data Differential expression values are presented as ratio of intensities between JNJ-26481585 supplier diabetic or diabetic-treated and control samples. Expression data were omitted in the regions where no signal was present or if the signal was just above local background or derived from,40% of the area of the printed spot. After normalization of the intensities, the data were filtered to exclude spots with intensities less than twice the background in either channel and finally, only spots with a normalized ratio greater than 1.5 were considered, since a minimum of 1.4fold change in differential expression can be accurately detected. However, only data with 2 fold-change or higher cutoff values are presented in these studies. Using the processed data we then performed in depth data analysis with the Genespring software. The significantly regulated genes were grouped into functional categories based on annotation by Gene Ontology, pathway analysis and PIR keywords using Genespring and DAVID version 2007. Animals Five-week-old male diabetic Otsuka Long-Evans Tokushima Fatty and normal male Long-Evans Tokushima Otsuka rats were obtained from Tokushima Research Institute, Otsuka Pharmaceutical Company. The OLETF is an established model of spontaneous non-insulindependent ty
