Absolute qpcr sybr green low rox mix

Total RNA from 4 × 10⁶ cells was isolated using the High Pure RNA isolation Kit (Roche, Mannheim, Germany), cDNA was synthesized from equal amounts of RNA using the first strand cDNA kit from Thermo Scientific (Waltham, MA USA). Quantitative real–time RT-PCR was performed using ABsoluteTM QPCR SYBR^®Green Low ROX Mix (Thermo Scientific, Waltham, MA, USA). Relative expression was calculated by normalization to β–Actin mRNA expression levels as 2-ΔCt. All primers were synthesized by Eurofins MWG Operon (Ebersberg, Germany) (Table 1).

4 × 10⁶ cells were harvested and subsequently total RNA was isolated using the High Pure RNA Isolation Kit (Roche, Mannheim, Germany). The first strand cDNA kit by Thermo Scientific (Waltham, MA, USA) was used for the synthesis of cDNA from equal amounts of RNA. ABsoluteTM qPCR SYBR^®Green Low ROX Mix (Thermo Scientific, Waltham, MA, USA) was used to perform quantitative real-time PCR. For the calculation of relative expression, normalization to β-Actin mRNA expression levels as 2-ΔCt was done. All primers were synthesized by Eurofins MWG Operon (Ebersberg, Germany). Human β-Actin, sense, 5′-aga gct acg agc tgc ctg ac-3′, antisense, 5′-agc act gtg ttg gcg tac ag-3′, human IDO, sense, 5′-tta gag tca aat ccc tca gtc c-3′, antisense, 5′-ttt gca gat ggt agc tcc tc-3′, human ADA, sense, 5′-gac ctg gct gga gat gag ac-3′, antisense, 5′-gtc ttc cag ggt gtg gta gc-3′, human ENTPD1 (CD39), sense, 5′-agt tct gtg ctc agc ctt gg-3′, antisense, 5′-ttg cag aag gag gga gag aa-3′, human NT5E (CD73), sense, 5′-ggg agg aca ctc caa cac at-3′, antisense, 5′-agg cct gga cta cag gaa cc-3′, human S100A8, sense, 5′-atg ccg tct aca ggg atg ac-3′, antisense, 5′-acg ccc atc ttt atc acc ag-3′, S100A9 sense, 5′-tca tca aca cct tcc acc aa-3′, antisense, 5′-gtg tcc agg tcc tcc atg at-3′.