*4.6. Quantitative Real-Time PCR and Conventional PCR*

Total RNA was isolated using the RNeasy Mini kit or RNeasy Micro kit (Qiagen, Venlo, The Netherlands) according to the manufacturer's instructions, including a DNAse step to remove genomic DNA. Subsequently cDNA was prepared using the RevertAid H Minus First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Landsmeer, The Netherlands). Quantitative PCR was performed using either Taqman® Universal PCR master mix combined with Taqman assays or SYBR® Green PCR master mix (all from Applied Biosystems, Life Technologies, Zwijndrecht, The Netherlands) combined with in house designed primers (Thermo Fisher Scientific, Landsmeer, The Netherlands). Taqman assays and primer sequences are described in Table S1. For detection we used a StepOnePlus™ Real-Time PCR System or the QuantStudio 3 (Applied Biosystems, Life Technologies, Zwijndrecht, The Netherlands). Values for each target gene were normalized by the expression level of 18S RNA. An arbitrary calibrator sample was used to correct for inter-plate differences. For calculating the relative quantity (RQ) the delta-delta Ct method was used for Taqman assays and a standard curve method was applied for SYBR green assays. An arbitrary calibrator sample was used to correct for inter-plate differences. The fold induction was calculated using the following formula: (RQ stimulated / RQ unstimulated).

For conventional PCR we used the GoTaq DNA, GoTaq green reaction buffer and dNTPs (Promega, Leiden, The Netherlands), and the Biometra T-gradient Thermoblock (Analytic Jena, Jena, Germany) with the following program: denaturation step 3 min at 95 ◦C, then cycle of 40 times of 30 s 95 ◦C followed by 30 s at 61 ◦C and elongation for 45 s at 72 ◦C and finally 2 min at 72 ◦C. As positive controls we used an in house made arbitrary RNA sample containing a mixture of RNA isolated from all human tissues (kindly provided by Dr. Huitinga from The Netherlands Institute for Neuroscience) as well as RNA from human thymus tissue (kindly provided by the Pathology department of the AMC, Amsterdam, The Netherlands). Samples were loaded on a 1.5% agarose gel (Invitrogen, Landsmeer, The Netherlands) and mRNA expression was visualized using gel imager Gene Flash (Syngene, Amsterdam, the Netherlands).
