Fastgene ic green 2 qpcr universal mix

Quantitative PCR (qPCR) amplification was performed using the SYBR green method in a 20 μL reaction mixture with the FastGene^® IC Green 2× qPCR Universal Mix (Nippon Genetics Europe, Düren, Germany) following the manufacturer’s instructions. For each gene a specific primer pair was used (Table 2). The qPCR reactions were carried out in a 36 well rotor using Rotor-Gene Q 5plex (Qiagen Inc., Hilden, Germany). The amplification was performed according to the following protocol: 95 °C for 2 min followed by 40 cycles of 95 ℃ for 5 s and annealing temperatures for 30 s. Quantification of gene expression levels was performed using the 2^−ΔΔCT method as described in our previous works [27 (link),76 (link),77 (link)]. β-actin was used as an internal control gene, and the median value of the NI group was used as a calibrator.

Reverse transcription-quantitative PCR (RT-qPCR) was performed to reveal the impact of rutin on the P. aeruginosa IBRS P001 quorum sensing (QS) regulatory genes expression. Primers used for the RT-qPCR analysis are listed in Table 4. The total RNA was extracted from the P. aeruginosa IBRS P001 test strain (grown for 10 h, 37 °C, aerobically) cultivated in Mueller-Hinton medium and supplemented with rutin (0.250 mg/mL, final concentration) or without it by an RNeasy Mini Kit (Qiagen, Hilden, Germany). The same volume of the ethanol (solvent) was added in the positive control. The total RNA was then treated with DNase I using an Ambion DNA-freeTM Kit (ThermoFisher, Waltham, MA, USA) and reverse transcribed by a Rever-tAid RT Reverse transcription Kit (ThermoFisher, Waltham, MA, USA) according to the manufacturer’s instructions. Further amplification was achieved with FastGene IC Green 2× qPCR Universal Mix (Nippon Genetics, Dueren, Germany) in a 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). Obtained data were normalized against the ribosomal gene rpsL as an internal control following the 2^−ΔΔCT method [73 (link)]. Experiments were done in triplicate.

The cDNA synthesis was executed starting from 500 ng of each RNA sample (i.e., exposed and not exposed to the EO) using PrimeScript™ RT reagent Kit (RR037A, Takara Bio Inc., Shiga, Japan), according to the manufacturer’s instructions. Each cDNA template was then used as substrate in qPCR prepared using the FastGene^® IC Green 2 × qPCR Universal Mix (LS4005, NIPPON Genetics EUROPE GmbH). Four virulence genes of L. monocytogenes were selected (hly, inlA, inlB and prfA) for this analysis, which was done as previously described [28 (link)]. Two reference (internal control) genes (tuf, gap) were included in each assay for the normalization of the qPCR data. Each experiment was repeated three times starting from independent bacterial cultures and each reaction was performed in triplicate.