Maxima probe rox qpcr master mix

To evaluate mRNA expression levels of Nrf2 and its target gene Nqo1, the Hippocampus and Cortex of mice between 8 and 12 weeks old were isolated and collected in RNAlater (Invitrogen). RNA extraction was performed using TRIzol (Invitrogen) and 500 ng RNA was synthesized to cDNA using the Maxima Reverse Transcriptase (Thermo Fisher).
For each quantitative real-time PCR (qPCR) reaction 40 ng cDNA was combined with Maxima Probe Rox qPCR Mastermix (Thermo Fisher) supplemented with adequate Taqman primers (Thermo Fisher) and 18S rRNA (Thermo Fisher) as an endogenous control using the StepOnePlus Real-Time PCR System (Applied Biosystems). Analysis was conducted with StepOne software v2.1. The following Taqman primers were used: Nrf2 (Mm00477784_m1) and Nqo1 (Mm01253561_m1).
Data were analyzed using the ∆∆CT method followed by relative quantification (2^−∆∆CT).

Coxiella burnetii was quantified by using real-time PCR (qPCR) targeting the isocitrate dehydrogenase encoding gene icd [48 (link), 49 ]. C. burnetii DNA was extracted by resuspending bacteria in phosphate-buffered saline (PBS) (BioWhittaker, Lonza, Walkersville, MD, USA) pH 7.4 and heat inactivated at 110 °C for 15 min. All qPCR reactions were performed in a 25 µl reaction mix using Maxima Probe/ROX qPCR Master Mix (ThermoFisher Scientific, Waltham, MA, USA), 300 nM each primer (icd-439 F and icd-514R), 100 nM probe (icd-464TM) and 2 µl of plasmid standard or sample [49 ]. The qPCR was carried out on a Mx3000P QPCR Instrument (Agilent Technologies, Santa Clara, CA, USA) using the following cycling conditions: 2 min at 50 °C, 10 min at 95 °C, followed by 45 cycles of 15 s at 95 °C and 30 s at 60 °C. Data collection and analysis was carried out using the Mx Pro4 software.

Following IFN-γ/LPS (M1) or IL-4/IL-13 (M2) stimulation, activated macrophages were lysed at 0, 24, 48, and 72 hr. RNA was extracted according to the manufacturer's instructions using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Genomic DNA contamination was removed with the RNA-Free DNase Set (Qiagen), and nucleic acid quantification was conducted using a Qubit 2.0 fluorometer (Life Technologies, Waltham, MA, USA). For cDNA synthesis, 30 ng/μL of RNA was utilized for RT-PCR with the High-capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). Gene expression was evaluated by quantitative real-time PCR (qPCR) and analyzed using the ΔΔCt method with TaqMan probes targeting TIM3 (Hs00958618_m1), GAL9 (Hs01088490_m1), BAT3 (Hs00190383_m1), and ADAM10 (Hs00153853_m1) genes. Concurrently, 18S ribosomal RNA gene (Hs03928990_g1) and ACTB (β-actin) (Hs01060665_g1) were employed as endogenous controls. Individual reactions were prepared using the Maxima Probe/ROX qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) and run under the following thermal cycling conditions: 95°C for 10 min, followed by 40 cycles of 60°C for 1 min and 95°C for 15 s in the Step One Plus Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). Unstimulated macrophages served as the reference condition (where RQ = 2^−ΔΔCT = 1).

Lung tissues were mechanically homogenized and total lung RNA purified with RNeasy Kit (Qiagen), adding a DNase I treatment step. cDNA was synthesized using the Maxima H Minus First Strand cDNA Synthesis Kit (ThermoFisher Scientific). The RT-PCR reaction was set up in duplicate with the Maxima Probe/ROX qPCR Master Mix (ThermoFisher Scientific) using commercial TaqMan primers for IFITM1 (Mm00850040_g1), IFITM3 (Mm00847057_s1), and the endogenous control GAPDH (Mm99999915_g1) and run in a Step One Real-Time PCR System (ThermoFisher Scientific) following the manufacturer's protocol. The double delta Ct method was used to quantify the relative mRNA expression of target genes.

In order to determine the presence or absence of C. jejuni in stool or mucosal tissue of patients, DNA was extracted and further analyzed using multiplex qPCR. For the extraction of DNA from stool samples, the QIAamp DNA stool mini kit (Qiagen, Hilden, Germany) was used, following the manufacturer’s instructions for the protocol to isolate DNA from stool for pathogen detection. DNA extraction from mucosal samples was carried out using the DNeasy Blood & Tissue Kit (Qiagen) and following the protocol for the purification of total DNA from animal tissues, according to the manufacturer’s instructions. Following extraction, a qPCR was performed, based on C. jejuni-specific mapA gene, using primers and probe described by [9 (link)]. Briefly, each reaction had a volume of 25 µL, containing 1 × Maxima Probe/ROX qPCR Master Mix (Thermo Scientific, Schwerte, Germany), 375 nM of each primer, 100 nM of probe labeled with FAM and quencher BHQ-2 (all Metabion, Planegg, Germany), 5 µL DNA template as well as 5.5 µL sterile water. The qPCR was performed using a CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA) with amplification parameters of 50 °C for 2 min and 95 °C for 10 min, as well as 45 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. Each sample was run in a technical duplicate, with positive and negative samples for comparison.

Total mRNA was obtained from microdissected mouse brain tissue using Dynabeads mRNA DIRECT Micro Kit according to the manufacturer’s (Life Technologies) instructions. cDNA was synthesized from purified mRNA by reverse transcription using the RevertAidH Minus Strand cDNA Synthesis Kit (Fermentas) and compromised oligo dT primers according to the manufacture’s manual. cDNA samples were stored at −20°C. For quantitative real time PCR the Maxima Probe/Rox qPCR Master Mix (Thermo Fischer) together with Taqman gene expression assays (Applied Biosystem) was used according to the following protocol: experiments were performed in triplicates on an ABI Prism 9700HT system (PE Applied Biosystems, Foster City, CA, USA). Gene expression was analyzed as relative gene expression in comparison to the internal reference gene synaptophysin. Therefore gene expression was calculated as 2-∆ct (D cycle threshold value (ct) = ct of the analyzed gene − ct synaptophysin).

RNA was extracted from tissue preserved in RNAlater^® using RNeasy Kits from Qiagen (74104,74704) and treated with RNase-Free DNase (79254, Qiagen). To check for RNA quality, random samples were chosen and analyzed with Agilent RNA 6000 Pico Kit on a 2100 Bioanalyzer from Agilent Technologies, Waldbronn, Germany, according to manufacturer’s instructions. All samples had a RIN value from 9.40–10.0. cDNA was prepared with RevertAid H minus Reverse Transcriptase and Random hexamer primer (Thermo Fischer Scientific) in a ThermoCycler from Biometra. TaqMan assays were from ThermoFischer (Table 1). Gene expression was quantified by real time PCR using Maxima^™ Probe/ROX qPCR master mix (Thermo Scientific) and the 7900HT Fast Real-Time PCR System, Applied Biosystems, with the software SDS 2.4. Relative gene expression was calculated according to the 2^-ΔΔCT method with Rn18S as the housekeeping gene.