The tubes were placed into a FastPrep (Bio 101) homogenizer and agitated twice at 6 m/s for 40 s. with 1 min-interval on ice. The next steps were performed according to manufacturer’s instructions. Finally, RNA samples were dissolved in 30 μl of RNase-free water. RNA integrity was tested with electrophoresis on 1% agarose gel. RNA quantification was performed measuring the absorbance at 260 nm. Nucleic acid purity was assessed measuring A260/A280 ratio (acceptable ratio was between 1.8 and 2.0). cDNA synthesis Reverse transcription was performed with the use of commercially available QuantiTect Reverse Transcription kit (QIAgen,

Hamburg, Germany). Firstly, 100 ng of total RNA was incubated with 2 μl of Wipeout buffer (QIAgen, Hamburg, Germany), containing RNase-free DNase, for

mTOR cancer 5 min. at 42°C. cDNA synthesis reaction was performed in a final volume of 20 μl, containing 100 ng of total RNA, 50 ng of random hexamer primers and the QuantiTect Reverse Transcriptase in RT buffer (QIAgen, Hamburg, Germany) according to the manufacturer’s instructions for the first-strand cDNA synthesis. Quantitative real-time PCR conditions The expression level of sodA and sodM genes were quantified using real-time RT-PCR (LightCycler® FastStart DNA Master SYBR Green I; Roche Diagnostics). Two μl of cDNA were subjected to amplification in a 20-μl volume containing 5 μM concentration of each primer (Table 3), 3 mM of MgCl2 and 2 μl of ready-to-use Light Cycler® DNA Master SYBR Green I (Roche Diagnostics). Pre-incubation step (95°C for 10 min.) was initially Selleckchem AZD5153 performed to activate FastStart DNA polymerase and to denature the template DNA. The following cycling conditions were used in the reaction: amplification and quantification program repeated 50 times

(95°C for 5 s, 66°C for 15 s and 10 s extension at 72°C with a single fluorescence measurement), melting curve program (65-95°C with a heating rate of 0.2°C per second and a continuous fluorescence measurement) and finally a cooling step to 40°C. Specificity of the PCR products was confirmed by analysis of the dissociation (-)-p-Bromotetramisole Oxalate curves. Expression levels of sodA and sodM genes were measured using an absolute quantification method that allows to determine the exact copy concentration of target gene by relating the Ct value to a standard curve. Ct value is defined as the point at which the fluorescence rises appreciably above the background fluorescence. Standard curve was constructed by amplifying known amounts of target DNA. Standard curves for sodA and sodM genes were generated using serial dilutions of a standard sample (calibrator): 1×, 0.5×, 0.2×, 0.1×. As a calibrator, https://www.selleckchem.com/products/CX-6258.html genomic DNA extracted from RN6390 strain (12.34 ng/μl) was used. In the case of sodA transcript quantification, amplification of sodA gene fragment was used, and similarly, to quantify sodM transcript level, sodM gene fragment from genomic DNA was used as calibrator.