Rt2 profiler pcr array human oxidative stress plus

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)].

Venous blood (2.5 mL) was collected in PAXgene Blood RNA Tubes (Qiagen) and RNA was isolated with PAXgene blood RNA kit (Qiagen) according to the manufacturer’s protocol. RNA quantification and quality control was performed using Nanodrop 2000 spectrophotometer (Thermo Scientific). RNA (400 ng) was reverse transcribed with the RT2 First Strand Kit (Qiagen). The expression of 84 key genes involved in redox responses and of 84 genes related to inflammatory processes was evaluated with RT2 Profiler™ PCR Array Human Oxidative Stress Plus (PAHS-065Y, Qiagen,
Extended data, Table S1 A)
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and RT
² Profiler™ PCR Array Human NF-κB Signalling Pathway (PAHS-025Z, Qiagen,
Extended data, Table S1 B).
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The SYBR Green chemistry on an ABI-7500 fast instrument (Thermo Fisher Scientific) was applied. The expression level of each transcript was normalized against the geometric mean of two housekeeping genes (
HPRT1 and
RPLP0) whose stability in blood was previously reported.
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Gene expression levels were calculated as 2
^−ΔCT values.