Qtower 2.0 thermal cycler

LLC cells were seeded into 60 mm Petri dish and incubated for 24 h (70–80% confluency), then treated with studied compounds for another 24 h. Total RNA was isolated using a commercial column-based Direct-zol RNA kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s recommendations. Commercial kit M09 (Ukrainian Genetic Technologies, Kyiv, Ukraine) was used for cDNA synthesis from 1 µg of total RNA per reaction. Real-time PCR was performed on qTOWER 2.0 thermal cycler (Analytik Jena, Jena, Germany) with SYBR Green-based kit M02 (Ukrainian Genetic Technologies, Kyiv, Ukraine). B2m was used for normalization, and ΔΔCt method—for quantification. Primers sequences are presented in Table 1.

P. polymyxa was activated overnight in TSB medium to determine the effects of xylose on the predicted operon. The latter had the overlapping promoters PxylR and PxylAB to regulate the transcription and the expression of XylA and XylB. The cells were incubated for another 12 h after 5% cultures were transferred to TSB medium, following which 2% xylose, glucose, or water was added. Each culture was sampled every 4 h. The mRNA was extracted with a Simply P Total RNA Extraction Kit (BSC52S1; BIOER, Hangzhou, China), and the cDNA was generated and amplified with a Prime Script RT Kit (TaKaRa Bio Inc., Kusatsu, Shiga, Japan). The genes xylR, xylA, and xylB were amplified with xR-F/xR-R, xA-F/xA-R, and xB-F/xB-r, respectively. The 16S housekeeping/internal reference gene was amplified with 16sF/R. The reaction conditions of the RT-qPCR were determined using a Universal YSBR qPCR Master Mix (Vazyme Biotech, Nanjing, China). The RT-qPCR was performed in a qTOWER2.0 thermal cycler (Analytik Jena, Jena, Germany).