Fermentas maxima sybr green rox qpcr master mix

For both human PBMCs and mouse samples, total RNA was isolated using TRIzol reagent (Life Technologies), and treated with DNase (Ambion/Life Technologies AM1906) after extraction. Only total RNA extracts with an OD260/OD280 ratio above 1.96, indicating relatively pure RNA, were processed for RT-PCR, the remainder undergoing re-extraction [39 (link)]. Total RNA was used to prepare cDNA via the Applied Biosystems High Capacity Archive Kit (#4368814). For detection and measurement of expression, Fermentas Maxima SYBR Green/ROX qPCR Master Mix (#K0222) was used. PCR mixtures were run on a Thermo Scientific PikoReal real-time PCR System using the following conditions: 95ᵒC for 10 minutes, followed by 40 cycles of 95ᵒC for 30 seconds, 60ᵒC for one minute and 72ᵒC for one minute. Cycle threshold (CT) value was used for relative quantification. For human PBMC samples, all values were normalized to three housekeeping genes, GAPDH, TFRC and b-actin using a geometric mean, and run in triplicate [40 ]. Mouse mRNA samples were normalized to b-actin. Primer sequences are listed in S1 Table, with human primers (h) used in PBMC samples and mouse (m) primers used in mouse brain extracts.