Kapa2g fast ready mix

Total RNA was isolated from the SCP-1 treated cells using Trifast (Peqlab, Erlangen, Germany) according to the manufacturer´s protocol and quantified using a spectrophotometer (Omega plate reader, BMG Labtech GmbH, Germany). cDNA was synthesized using the First Strand cDNA Synthesis Kit from 2500 ng total RNA (Fermentas St, Leon-Rot, Germany). Afterwards, semi-quantitative RT-PCR was performed from the 10 ng cDNA template using KAPA2G Fast Ready Mix (Peqlab, Erlangen, Germany). Primers and PCR conditions were previously optimized with increasing amounts of cDNA, in order to analyzed the PCR product obtained from the logarithmic phase. Primer sequences and PCR conditions are shown in Table 2. GAPDH was used as an internal control for normalization. PCR products were resolved using a 1.5% agarose gel, with ethidium bromide for visualization. Densitometric analysis was performed using the ImageJ software.

Total RNA was isolated using the Trifast reagent (Peqlab, Erlangen, Germany) as indicated by the manufacturer; cDNA sysnthesis was performed from 2 µg total RNA using the First Strand cDNA Synthesis Kit from Fermentas (ThermoScientific, Karlsruhe, Germany). Gene expression changes were investigated by semi-quantitative RT-PCR using the KAPA2G Fast Ready Mix from Peqlab. PCR conditions are summarized in Table 2. PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide (geldoc, INTAS, Göttingen, Germany). Individual samples were run twice (n = 2) to reduce the variations caused by small loading differences. The signal intensities were quantified using the ImageJ software (NIH, version 1.49m) [31 (link)].

Trifast reagent (Peqlab, Erlangen, Germany) was used to isolate total RNA. Expression of oxidative stress related genes was screened with the RT² Profiler PCR Array human oxidative stress plus (Qiagen, Hilden, Germany). The array was performed with 2 pools (each N = 8 donors) of RNA samples to minimize the donor dependent variations. RNA purification, cDNA synthesis and the array itself were performed as indicated by the manufacturer, using the advised products from Qiagen. Semi-quantitative RT-PCR was done to confirm gene expression changes of the individual samples. RT-PCR was performed with the KAPA2G Fast Ready Mix from Peqlab, using the primers for NOX4 (NM_016931.4) forward: 5′-CGGGCTTCCACTCAGTCTTT-3′ and reverse: 5′-TCCTAGCCCCAACATCTGGT-3′ and GAPDH (glycerinaldehyd-3-phosphat-dehydrogenase/NM_002046.4) forward: 5′-GTCAGTGGTGGACCTGACCT-3’ and reverse: 5′-AGGGGTCTACAT GGCAACTG-3′. PCR products, separated by agarose gel electrophoresis, were visualized by ethidium bromide (geldoc/INTAS, Göttingen, Germany). Each sample was loaded twice (n = 2) to minimize loading differences. Signal intensities were quantified using the ImageJ software [42 (link)].