Sensimix sybr no rox

Total RNA was prepared from cultured cells using the ReliaPrep™ RNA Cell Miniprep System kit from Promega according to the manufacturer’s protocol. Two micrograms of RNA was used for reverse transcription with Rotor-Gene SYBR Green RT-PCR(QIAGEN) or The SensiMix™ SYBR® No-ROX(BioLine) protocols for Real TimeOne-Step RT-PCR using the Real Time Rotor-GeneRG-3000™ light cycler from Corbett Research using primers listed in Supplementary Table 4.

Total RNA was prepared from cultured cells using the ReliaPrep™ RNA Cell Miniprep System kit from Promega according to the manufacturer’s protocol. Two micrograms of RNA was used for reverse transcription with Rotor-Gene SYBR Green RT-PCR (QIAGEN) or The SensiMix™ SYBR® No-ROX (BioLine) protocols for Real TimeOne-Step RT-PCR using the Real Time Rotor-GeneRG-3000™ light cycler from Corbett Research using primers listed in Supplementary Table IV.

Total RNA was extracted from FFPE mouse brain tissues using the ReliaPrep™ FFPE Total RNA Miniprep System Kit (Promega) and following manufacturers’ instructions. The concentration of RNA was measured using a NanoPhotometer P300 (Implen). Samples were used for cDNA synthesis using the Superscript™ III First‐Strand Synthesis System for RT‐PCR (Invitrogen), following manufacturer's instructions. Quantitative real‐time PCR was performed in a LightCycler^® 480 Multiwell Plate 96 (Roche) using SensiMix SYBR No‐Rox (Bioline), in triplicates, as previously described (Althubiti et al., 2016). For the primers used, see Table S4. All primers were supplied by Eurofins Scientific. A melting curve was used to prove the specificity of the primers at the end of the PCR run. Quantification was done with the ∆∆C_t method. Results were analysed on Microsoft Excel, and graphs plotted using GraphPad Prism 7.0 Software.

Total RNA was extracted using QIAzol Lysis Reagent and isolated using RNAeasy mini kit (both from QIAGEN). cDNA synthesis was performed using the ProtoScript II First Strand cDNA Synthesis Kit (NEB). Expression levels of target genes were determined by RT-PCR using SensiMix SYBR No-Rox (Bioline) with the primers shown in Supplementary Table 1. Reactions were run on an MX3000P real-time thermal cycler (Agilent Technologies) and Ct values determined using MxPro qPCR software. Relative expression levels were calculated using the 2^-ΔCt method after normalising to the expression levels of the housekeeping gene, Gapdh. Fold change levels were calculated using the 2^-ΔΔCt method after normalising to the untreated control.

Quantitative real-time PCR (qPCR) of nirK, nirS and 16S rRNA genes was carried out using a Lightcycler 480 II (Roche, Applied Science). Standard curves were prepared from serial dilutions of linearized plasmid with the nirK gene from Alcaligenes faecalis LMG 1229^T, nirS gene from Paracoccus sp. R-24615 and 16S rRNA gene from Flavobacterium swingsii LMG 25510, containing between 10⁹ to 10¹ target gene copies calculated directly from the concentration of the extracted plasmid. DNA concentrations were determined using the Nanodrop 2000 spectrophotometer (Thermo, Scientific). The qPCR assays were carried out in a 20 μl reaction volume composed of SensiMix SYBR No-ROX (Bioline GmbH, Luckenwalde, Germany), 0.4 μM of each primer, 2.5 μl of template DNA (10 ng/μl), 0.1 mg BSA (not used in the nirS assay) and sterilized milliQ water. The same primers as for pyrosequencing were used and the thermal protocol can be found in S2 Table. All reactions per sample were performed in triplicate. Agarose gel electrophoresis, melting curve analysis, cloning and sequencing of the obtained amplicons indicated that the amplification was specific. PCR inhibition was determined by spiking sediment DNA with a known amount of standard DNA and corrected for according to Zaprasis et al. [65 (link)].