Superscript reverse transcriptase kit with oligo dt primers

mRNA was isolated from the brains of the indicated groups of mice using an RNAeasy kit (Qiagen, Germany) (n = 4 per group). The SuperScript reverse transcriptase kit with oligo (dT) primers (Invitrogen, Canada) was used to transcribe the mRNA into cDNA. Quantitative-PCR for interferon-γ (IFN-γ), tumor necrosis factor (TNF), chemokine (C-X-C motif) ligand 3 (CXCL3), CXCL9, CXCL10, CXCL11, lymphotoxin α (LTα), LTβ and hypoxanthine phosphoribosyltransferase (HPRT) was performed using cDNA from olfactory bulb, cortex, and brainstem regions, respectively, of uninfected (day 0) and infected mice (day 5 and 7 p.i.) with and without pyrimethamine-treated mice employing the respective Taqman gene expression assay (Applied Biosystems, Germany). To determine the parasite load in the different regions of the brain, q-PCR was performed for P. berghei ANKA cytochrome B (PBANKA_MIT01900)^{72 (link)} and HPRT (Eurofins Genomics, Germany) using Light cycler 480 SyBr Green I master mix (Roche, Germany). Amplification was performed with a Light cycler 480 (Roche, Germany). Quantitation was performed using the Light cycler software SDS (version 5.1; Applied Biosystems, Germany), according to the ddC_T threshold cycle method with HPRT as the housekeeping gene^{73 (link)}. Data are depicted as the increase in the level of mRNA expression in infected mice over uninfected control mice.

Isolation of mRNA from the brains of uninfected and PbA-infected mice was performed with an RNAeasy kit (Qiagen, Hilden, Germany). The SuperScript reverse transcriptase kit with oligo (dT) primers (Invitrogen) was used to transcribe mRNA into cDNA. Quantitative RT-PCR for IFN-γ, perforin, granzyme B, TNF, IL-6, CXCL-9, CXCL-10, LT-α, and hypoxanthine phosphoribosyltransferase (HPRT) was performed with cDNA from C57BL/6 WT and C57BL/6 Cyld^−/− mice and the respective Taqman gene expression assay (Applied Biosystems, Darmstadt, Germany). Amplification was performed with a GeneAmp 5700 sequence detection system (Applied Biosystems). Quantitation was performed with the sequence detector software SDS (version 2.1; Applied Biosystems), according to the ΔΔC_T threshold cycle method with HPRT as the housekeeping gene (52 (link)). Data are expressed as the increase in the level of mRNA expression in infected mice over that in uninfected controls of the respective mouse strain. All primers and probes were obtained from Applied Biosystems.

Total mRNA was isolated using the Qiashredder and RNeasy Mini Kit from Qiagen according to the manufacturer’s instructions. The SuperScript reverse transcriptase kit with oligo (dT) primers (Invitrogen) was used to generate cDNA. Quantitative RT-PCR for klotho and β-actin (Applied Biosystems) was performed on the Lightcycler 480 system (Roche). The ratio between the respective gene and corresponding β-actin was calculated per sample according to the ∆∆ cycle threshold method [36 (link)].