Ng dart rt pcr reagents

The expression level of genes connected with DNA repair and oxidative stress response was evaluated with quantitative real-time PCR (qRT-PCR) method. The A375 cells were seeded in 6-well culture plates at a density 1 × 10⁵ cells/mL and after 70–80% confluency achievement the cells were treated with CuT1, Cu10, CuT16 in concentrations corresponding to IC₅₀ values or DMSO as vehicle in control cultures. After 24h of incubation total RNA was isolated using the Syngen Blood/Cell RNA Mini Kit (Syngen Biotech, Wroclaw, Poland). All samples were transcribed using NG dART RT-PCR reagents (EURx, Gdansk, Poland) according to the manufacturer’s instructions. The relative mRNA expression level was determined by relative quantification method (ΔΔCt) using 18S ribosomal N5 (RNA18SN5) and beta-actin (ACTB) as endogenous controls. The reference genes were selected on the basis of our preliminary studies, where RNA18SN5 and ACTB remained unaffected by the experimental conditions. The PCR was conducted in triplicate using Applied Biosystems^® 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) and SG qPCR Master Mix (2×) (EURx, Gdansk, Poland) in accordance with the manufacturer’s protocol. Data was presented and analysed as RQ values (RQ = 2^−ΔΔCt). The genes and sequences of used primers were listed in Table 5.

All samples of good quality (OD 260/280 ratios approximately 2.0) were reverse transcribed using random primers and NG dART RT-PCR reagents (EURx, Gdańsk, Poland) as described by the manufacturer. To analyze the correlation of diabetes with the cognitive function, the following primers were used: Arc, Bdnf, Egr1 [see Table 1]. The analysis of the genes’ expression levels was performed by real-time PCR method using the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and Fast Probe qPCR Master Mix (2x), plus ROX Solution (EURx, Poland). Briefly, the reaction mixture contained 10 µL of Fast Probe qPCR Master Mix (2x), 9 µL of RNase-free water, ROX Solution (50 nM), and 0.5 µM of gene-specific TaqMan probe (Applied Biosystems, Foster City, CA, USA). The reactions were performed as followed: 95 °C for 3 min, 40 cycles: 95 °C for 10 s and 60 °C for 30 s. The data quality screen based on amplification, Tm and Ct values was performed to remove any outlier data before ΔΔCt calculations and to determine fold change in mRNA levels. The data were presented as a mean RQ ± SEM value (RQ = 2−ΔΔCt).