Sensifast sybr no rox one step kit

For quantitative real time PCR at least five independent experiments were carried out. At first, RNA content was measured using Qubit RNA Assay Kit (Life technologies, Waltham, MA, USA). Quantitative real time PCR was performed using SensiFast TM SYBR/No-ROX One Step Kit (2 ng RNA; 10 pmol of each primer (Supplementary Table A); Bioline, Bochum, Germany) according to manufacturer’s protocol. β-Actin served as housekeeping gene. RNA reverse transcription, amplification and quantification was operated using qTower (Analytik Jena AG, Jena, Germany). For reverse transcription samples were heated up to 45 °C for 10 min followed by the activation of polymerase (95 °C, 2 min) and 40 cycles of denaturation (95 °C, 5 s), and annealing with elongation (varying temperature according to the primer, 20 s) in alternating order. Experiments were carried out in sets of three replicates, whose results were averaged and further mathematically processed using ΔΔ-CT method.

For gene expression analyses of purified alveolar macrophages, RNA was analyzed using the SensiFAST^TM SYBR^® No-ROX One-Step Kit (Bioline) according to the manufacturer’s instructions. Transcript levels were normalized to the housekeeping gene Actb. Following primer sequences were used: Actb, 5′-CTTCTTTGCAGCTCCTTCGT-3′ (forward) and 5′-TCCTTCTGACCCATTCCCAC-3′ (reverse); Msr1, 5′-GGAATAAGAGGTATTCCAGGTG-3′ (forward) and 5′-TTTGTCCTTTAGGTCCAGGAG-3′ (reverse); Fizz1, 5′-ACGAGTAAGCACAGGCAGTT-3′ (forward) and 5′-TGCCAATCCAGCTAACTATCCC-3′ (reverse).

The relative gene expression levels were determined at pH 5 to pH 8 in the presence and absence of GEN. Both E. coli strains were cultured in LB at pH 5 to pH 8 and total RNA was isolated after 8 h of culturing. The relative expression levels of the marR multiple antibiotic resistance regulator and the sdiA quorum sensing activator genes were determined by RT-qPCR. This involved the CFX96 Touch real-time PCR detection system (BioRad, Hercules, CA, USA), strictly following the manufacturer’s recommendations for the SensiFAST^TM SYBR No-ROX One-Step Kit (Bioline GmbH, Luckenwalde, Germany). Briefly, each well of the 96-well microtiter plate contained 20 µL as follows: 10 µL of the 2× SensiFAST^TM SYBR No-ROX One-Step Mix, 0.2 µL Reverse Transcriptase, 0.4 µL RiboSafe RNase Inhibitor, 5.4 µL Diethylpyrocarbonate (DEPC)-treated water, 500 nM of each primer and approximately 20 ng of total RNA in RNase-free water. Thermal cycling was initiated with a denaturation step of 5 min at 95 °C, followed by 40 cycles each of 10 s at 95 °C, 30 s at 57 °C, and 20 s at 72 °C. The relative quantities of the mRNA of each gene of interest were determined by ΔΔC_T method. Gene transcript levels were normalized against the E. coli housekeeping gene GAPDH measured in the same sample. The primers used in the assay shown in Table 7.

Real-time RT-PCR was performed with DNaseI (RQ1 RNase-free DNase, Promega)-digested total RNA samples which was subjected to reverse transcription and PCR amplification using the SensiFAST^TM SYBR No-ROX One-Step Kit (BIOLINE, Germany). SYBR Green I fluorescence was recorded on a DNA Engine Opticon (Bio-RAD, Germany). Per sample 100 ng total RNA were used and RPL13 (Gene ID: 5718254) as well as RACK1 (GeneID: 5723548) served as housekeeping genes. The following primers were used within the study: IFR1 (5′-ATGGCGACTAAGAAGCACAC-3′ and 5′-CGAAGCCTGCTCATTGTAGT-3′), RPL13 (5′-ATTCTTGCCGGGCAGCAGATTGTG-3′ and 5′-TTGCGCAGGAAG CGGTCATACTTC-3′) and RACK1 (5′-TCAACATCACCAGCAAGAAGG-3′ and 5′-CTGGGCATTTACAGGGAGTG-3′). Relative mRNA expression levels were calculated according to Pfaffl (Pfaffl, 2001 (link)).

Viral load was quantified in capsid gene copy number units using the qRT-PCR method essentially as described previously [14 (link)]. Reactions were performed in duplicate using the SensiFAST SYBR No-ROX One-Step kit (Bioline, Alexandra, NSW, Australia) on a BioRad CFX96/C1000 thermal cycler platform using the primers RHDV-RT2_fw 5’-ACCCAGTACGGCACRGGCTCCCAACCAC-3′ and RHDV-RT2_rv 5’-CTATCTCCATGAAACCAGATGCAAAGGT-3′ [15 (link)].

Reverse transcription quantitative PCR was carried out according to the protocol of Wolf et al. (2017a) (link) by use of the SensiFast SYBR No-Rox One-Step Kit (Bioline, London, United Kingdom) in 96 well lightcycler plates (Sarstedt, Nümbrecht, Germany) in a LightCycler 96 system of Roche (Mannheim, Germany) by use of the software LightCycler^® 96 SW 1.1 (Roche, Mannheim, Germany). The relative RNA amount was normalized on total RNA (100 ng) and calculated as 2^–ΔCq. ΔCq was calculated as the difference of the mean Cq in the mutant strain compared to the control strain. The primers are listed in Supplementary Table S2.