, USA). Reverse transcriptase

(RT) reactions buy Cyclosporin A utilized 10 ng of RNA sample, 50 nM of stem-loop RT primer, 1 × RT buffer and 0.25 mM each of dNTPs, 3.33 U/μl MultiScribe RT and 0.25 U/μl RNase inhibitor (all from the TaqMan MicroRNA Reverse Transcription kit of Applied Biosystems; 4366597). Reaction mixtures (15 μl) were incubated in a TGradient thermal cycler (Biometra) for 30 min at 16°C, 30 min at 42°C, 5 min at 85°C, and then held at 4°C. Real-time PCR was performed using the Applied Biosystems 7500 Sequence Detection System. The 20-μl PCR reaction mixture included 1.3 μl of RT product, 1 × TaqMan (NoUmpErase UNG) Universal PCR Master Mix, and 1 μl of primer and probe mix of the TaqMan MicroRNA Assay protocol (PE Applied Biosystems). Reactions were incubated in a 96-well optical plate at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 10 min. The threshold cycle data were determined using the default threshold settings. All real-time PCR reactions were run in triplicate and average threshold cycle (CT) and SD values were calculated. Data normalization and statistical analysis Expression data were normalized according to expression of the RNU6B

reference DNA (Assay No. 4373381; Applied Biosystems). Statistical differences between miRNA levels in RCCs and RP and differences in therapy response in relation to miRNA levels were buy AZD1480 evaluated using the nonparametric Mann-Whitney U test between 2 groups. Survival analyses were performed using the long-rank Omipalisib cell line test and Kaplan-Meier plots approach. All calculations were performed using Statistica software version 6.0 (StatSoft Inc., USA). Results

We identified gene expression levels of the studied miRNAs in 38 RCCs and 10 non-tumoral renal parenchyma (RP). Differences enough between the two groups were evaluated using the Mann-Whitney test and also by the Wilcoxon test for ten paired samples. Both methods identified highly significant differences between RCC and RP in the expression levels of the most studied miRNAs. Significance levels and medians of the relative expression values with their ranges defined by the 25th and 75th percentiles are presented in Table 2. The real-time PCR analysis indicated no significant difference between RCC and the RP in expression levels of miR-200b and miR-182. By contrast, the expression levels of miR-155, miR-210, miR-106a and miR-106b were significantly upregulated in the tumor compared to the RP. The most significant difference was seen for miR-210, for which the expression levels were more than 60 times higher in RCC tissue. Conversely, miR-141 and miR-200 were significantly downregulated in RCCs (Table 2). The most significant difference was observed in miR-141, with levels in RCCs approximately 15 times lower than in the RP.