Per patient, at least 6 sections were studied. In case of IHD, only vital cardiomyocytes were examined. Necrotic or fibrotic areas were excluded from examination. The mRNA was isolated from 20 frozen tissue sections (thickness 10 μm) of myocardial biopsies using Dynabeads Oligo(dT)25 (Invitrogen Dynal AS, Oslo, Norway). cDNA synthesis was performed using oligo-dT, random primers, and superscript-III. The primer/probe combinations used for Q-PCR were from Applied Biosystems (Foster City, CA, USA) either in a low-density array (LDA) or as single

tests (Taqman Gene Expression Assays: TGEA). The LDAs were used according the manufacturer’s instructions and for TGEAs per reaction 12.5 μl Taqman LY2157299 purchase selleck kinase inhibitor universal master mix (Applied Biosystems), 1.25 μl primer/probe, 6.25 μl milliQ was used and 5 μl cDNA sample was added. The Q-PCR reactions were carried out by the 7900 sequence detection system of Applied Biosystems. Thermal cycling comprised a denaturation step at 95°C for 10 min followed by 45 cycles of 95°C for 15 s and 60°C for 60 s. All experiments were performed in duplicate. For standardization, the expression of three endogenous control genes was tested in parallel. These three genes showed differences in expression level but the relative expression remained the same. The mean quantification cycle threshold (Cq) value

of all samples was 29.31±0.91 for HMBS, 19.35±1.05 for GAPDH, and 25.06±1.24 for PGK-1. We decided to use GAPDH for quantification as its expression level lies within the most reliable Ct range. To quantify the data, the comparative Cq method was used, resulting in relative mRNA quantities (RQ) [15]. The Q-PCR data were analyzed using the paired and unpaired t-test when appropriate (based on normal distribution tested by

the Kolmogorov–Smirnov test). All data were calculated with the statistical package of Prism 4.0 for Windows. A P value <.05 was considered statistically significant. 17-DMAG (Alvespimycin) HCl The fold change between pre- and post-LVAD gene expression for the differentially expressed genes was determined by calculating the RQ post/RQ pre ratio for each patient. Furthermore, the average ratio of all patients was determined. Fold change was Log2 (average ratio). To evaluate the location and expression of the different integrins in the cardiovascular system pre and post LVAD support, frozen tissue sections were stained by a conventional three-step immunoperoxidase staining and scored. The location of integrin-α5, -α6, -α7, -β1, and -β6 was established. The results, as summarized in Table 3 and Fig. 1, show that integrin-α6 is restricted to the capillaries and integrin-β1 to the membrane of cardiomyocytes. Integrin-α7 occurs in the cardiomyocyte membrane, the intercalated discs, and in the cytoplasm of the cardiomyocytes.