Reverse transcription from RNA to DNA was performed with a Multiscribe Reverse Transcriptase kit from Applied Biosystem at 25°C for 10 min, at 48°C for 30 min and at 94°C for 29 sec. The PCR was performed in triplicates of each sample in a volume of 25 μL in each well containing RNA, TaqMan Universal PCR MasterMix and a primer of the target, i.e., HIF-1α (Rn00577560_m1), TGF-β (Rn00572010_m1) and VEGF-A (Rn4331348), and a primer of the housekeeping gene, 18S (4319413), all purchased from Applied Biosystems. Each RT-PCR reaction ran at 50°C for 2 min, at 95°C

for 10 min and in 40 cycles changing between 95°C for 15 sec. and 60°C for 1.30 min [27]. PCR Data analysis Data was analyzed with the ABI Prism 7000 Sequence Detector Software from Applied Biosystems. The output of amplification was measured in the Selonsertib datasheet exponential phase of the reaction as the threshold cycle/Ct-value, which is defined as the cycle number at which amplification products are detected corresponding to the point where fluorescent intensity exceeds the background fluorescent intensity, which is 10 × the standard deviation of the baseline. The average of triplicates from each sample was used. The relative quantification of target gene was calculated using the formula: (1/2)Ct-target gene- Ct-housekeeping gene, which is described in the Users Bulletin 2,

1997 from Perkin-Elmer (Perkin-Elmer Cetus, Norwalk, CT, USA) [27]. Statistical selleck chemicals llc analysis Statistical analysis were performed by SPSS® 11.0 programs (SPSS Inc., Chicago, Illinois, USA). All data is expressed as mean ± SEM. Comparisons of data between groups were performed by non-parametric Kruskal-Wallis (ANOVA) test followed by the Mann-Whitney U-test. A p value < 0.05 was considered significant. Results Liver

parameters Blood samples showed a significant increase in ALAT in group IRI (334 ± 135 U/L), IPC (377 ± 104 U/L), IPO (1177 ± 379 U/L) and IPC+IPO (710 ± 199 U/L) compared to the control group (40 ± 2 U/L) (CG vs. IRI, IPC, IPO, and IPC+IPO, p = 0.01). No significant differences were found in ALAT between groups IRI, IPC, IPO and IPC+IPO. Alkaline phosphates and bilirubin were comparable between groups (Figure 2). Figure 2 Blood samples including ALAT (A), alkaline phosphatase (AP) (B) and bilirubin (C) levels. Samples 30 min after reperfusion in CG, Control group. IRI, 30 min of ischemia. IPC, ischemic mTOR target preconditioning + 30 min of MycoClean Mycoplasma Removal Kit ischemia. IPO, 30 min ischemia + ischemic postconditioning. IPC+IPO, ischemic preconditioning + 30 min of ischemia + ischemic postconditioning. * indicates p ≤ 0.01 compared to the control group. HIF-1α expression In the IRI group the expression of HIF-1α mRNA was significantly increased after 30 min of reperfusion compared to the control group (p ≤ 0.01). In the IPC group HIF-1α mRNA expression was significantly lower than the IRI group (p ≤ 0.01). In rats subjected to IPO there was a tendency towards lower HIF-1α mRNA expression compared to the IRI group (p = 0.