Rnaprep bacteria kit

RNA was prepared from triplicate cultures of the P. aeruginosa PAO1 nosZ-deletion mutant and wild-type strains grown in modified AB minimal media with 8.6 mM acetate as the electron donor and 7.2 mM NO₂⁻ as the electron acceptor. All solutions used during the RNA extraction process were prepared with DEPC-treated water (Adil). Cells were split into 50-ml conical tubes (BD Biosciences, San Jose, CA) and pelleted by centrifugation at 3,000 g for 15 min. RNA was then extracted with the RNAprep Bacteria kit (TianGen, China) and treated with DNA-free DNase (Ambion) according to the manufacturer’s instructions. All RNA samples were checked for integrity by agarose gel electrophoresis and had A₂₆₀/A₂₈₀ ratios of 1.8 to 2.2. In order to ensure that RNA samples were not contaminated with DNA, PCR amplification with primers targeting the 16S rRNA gene was conducted on RNA samples that had not undergone reverse transcription.

RAW264.7 cells were grown at 37°C with 5% CO₂ for 24 h in 12-well plates and infected with Listeria strains at a MOI of 100:1 for 1 h. Cells were washed and treated with DMEM containing 200 μg/mL gentamicin for 1 h to kill the extracellular bacteria, and then were kept incubated in medium containing 20 μg/mL gentamicin for additional 5 h.
Total RNA of bacteria from BHI broth or RAW264.7 cells was isolated using a RNAprep Bacteria Kit (TianGen, China), and was reverse transcripted to cDNA using the iScript™ gDNA Clear cDNA Synthesis Kit (Bio-Rad, USA). msv mRNA transcript abundance was normalized to rpoB mRNA transcript abundance. msv was amplified by primers msv-f : 5′-TGATCGTGTACGTGGAGCAG-3′ and msv-r: 5′-GGACCACCACTCATAGCACC-3′, LM- rpoB was amplified by primers LM- rpoB-f : 5′-AATCGGGGACAATGACT-3′ and LM- rpoB-r: 5′-GTGTGCGGAAACCTAC-3′, LI- rpoB was amplified with primers LI- rpoB-f : 5′-TCCGTTCAGAAAACTTAGCGGT-3′ and LI- rpoB-r: 5′-GCAGTTACAGCAGCACCAGAGT-3′. Quantification was performed on the CFX96 (Bio-Rad) with an initial 98°C for 2 min, followed by 40 cycles of denaturation at 98°C for 5 s, annealing and extension at 55°C for 5 s. Melting point analysis was performed to ensure the specificity of the amplification.