RNA was isolated from mice lung segments stored in RNAlater (Qiagen, Hilden, Germany), following the RNeasy mini kit protocol (QIAGEN) according to the manufacturer’s recommendations. RNA concentration was measured spectrophotometrically using the absorbance ratio of 260/280 nm. Gel electrophoresis was used to check the purity and integrity of the total RNA. Additionally, the fragmented bacterial RNA was analyzed using the Agilent 2100 Bioanalyzer System (Agilent Technologies, Waldbronn, Germany) following the manufacturer’s protocol. 1 μg isolated RNA from P. aeruginosa was used as template, with the first strand iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) for the reverse transcription for cDNA synthesis; conditions were 5 min at 25°C, 30 min at 42°C, and 5 min at 85°C. Quantitative real-time PCR (qRT-PCR) analysis of the expression of quorum sensing (QS) genes rhll, rhlR, lasl, lasR, pqsA and pqsR was carried out using the P. aeruginosa specific primers described [24] (Table C in S1 Text). All primers were purchased from IDT (IDT, San Jose, California, U.S.A). qRT- PCR was performed with the iQ5 cycler system (BioRad, Hercules, CA, USA) using 2 μl cDNA, 10 μl iTaq Universal SYBR Green Supermix, (BioRad), 6μl Molecular Grade Water (AppliChem) and 0.1 pmol/ml sense and antisense primers to a final volume of 20-μl on duplicate samples in 96-well plate. The thermal cycler profile involved a preliminary denaturation at 95°C for 1 min; followed by 40 cycles of denaturation at 95°C for 30 s; annealing at 52°C for 30 s; and extension for at 72°C 1 min.; followed by cooling at 4°C. Amplification was followed by a melting curve from 55°C to 95°C increasing of 0.5 each time for verification of the amplified product. The threshold was adjusted according to the amplification curves of all evaluated genes. No reverse transcriptase controls were prepared from total RNA and no template controls were prepared with Molecular Grade Water (AppliChem) in place of total RNA to indicate potential genomic DNA contamination in isolated bacterial RNA and contamination of reagents, respectively. Specificity was confirmed by electrophoretic analysis in 3% w/v agarose electrophoresis gel (5V/cm) and ethidium bromide staining (AppliChem). Analysis of relative gene expression was achieved according to the ΔΔ CT method [25]. The number of transcripts of QS genes in the lung was normalized to the respective number of bacteria in the lung at the time of sacrifice [26].

Additionally, each MDR P. aeruginosa isolate was incubated at a starting inoculum of 1 x 107 cfu/ml in tubes with Mueller-Hinton broth with and without 1mM of the H2S donor GYY4137 (Sigma-Aldrich) and with and without 1mM of the AOAA (Sigma-Aldrich). After 2 and 6 hours of incubation RNA was isolated from 1ml of each dilution, using the same methodology as described above. RNA was then used for measurement of expression of quorum sensing genes. An aliquot of 0.1ml of each dilution was diluted 1:10 into Mueller-Hinton broth (Becton Dickinson) six consecutive times; 0.1 ml of each dilution was plated onto MacConkey agar (Becton Dickinson). After incubation for 24 hours at 37°C, the number of viable colonies was counted. The results were expressed as log10 of colony forming units per ml (cfu/ml). The number of transcripts of QS genes was normalized to the respective bacterial load. The experiment was performed in duplicate.

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