Cfx96 equipment

Leukocyte total RNA was extracted using Trizol reagent (Life Technologies, Inc) according to the manufacturer’s instructions. RNA was quantified with a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies), and cDNA was synthesized from 1 μg ARN with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Cat. # 4368814).). Real-time PCR was performed in duplicate using CFX96 equipment (BioRad) and SYBR® Green PCR Master Mix, according to the manufacturer’s protocol. PPAR-α, PPAR-β, PPAR-γ, incretins receptor genes GLP-1R and GIPR, and reference genes PGK1 and YWHAZ were quantitated using the gene-specific primers (Additional file 1). cDNAs were amplified for 40 cycles consisting of 10 s of denaturation at 95 °C, 15 s of annealing temperature for each primer (Additional file 1), and 10 s of extension at 72 °C. Standard curves for all genes were generated using serial dilutions of pooled cDNAs from all samples. Relative mRNA expression was calculated with the method ΔΔCt. Data are shown as normalized ratios between target gene expression and geometric media of the two reference genes [56 (link)]. All expression assays were performed following the MIQE guidelines [57 (link)].

The nasal washes were processed using the Anyplex II RV16 kit (Seegene, Seoul, South Korea). The nucleic acid extraction was performed manually using the Ribo_spin GeneAll vRD kit (GeneAll Biotechnology, Seoul, South Korea); before extraction, an internal control of the Anyplex kit was added to each sample. The cDNA was synthesized from extracted RNA with the cDNA Synthesis Kit Premix (Seegene). Subsequently, real-time PCR was carried out in the CFX96 equipment (Bio-Rad) through the Anyplex II RV16 Detection kit, which uses TOCE technology. Amplified respiratory viruses were visualized using the Seegene Viewer software. The Anyplex RV16 kit has the capacity to detect 14 RNA viruses and 2 DNA viruses: respiratory syncytial virus types A and B; influenza virus types A and B; parainfluenza virus types 1–4; adenovirus; metapneumovirus; coronavirus OC43, 229E, and NL63; rhinovirus types A, B, and C; enterovirus; and bocavirus. Bacterial genetic material was detected in nasal washings using the Seeplex RV15 ACE detection kit (Seegene, Seoul Korea) with simultaneous detection with a multiplex PCR using the DPO technology. The kit detects Streptococcus pneumoniae, Haemophilus influenzae, Chlamydophila pneumoniae, Legionella pneumophila, Bordetella pertussis, and Mycoplasma pneumoniae.

The quantitative real-time RT-PCR with the rpsl gene as the reference was performed to monitor the expression changes of czcS, czcR, czcC and oprD genes when P. aeruginosa is stimulated by the Zn(II) at a micromolar level. The total RNA was extracted by traditional phenol-chloroform method and reverse transcribed by iScript cDNA Synthesis Kit (Bio-Rad). The cDNA samples were diluted for different folds and used as the templates in the PCR experiments. The real-time RT-PCR was performed on the Bio-Rad CFX96 equipment using the Ssofast SYBR Green Supermix (Bio-Rad). The experiments were performed at least three independent times with average results shown. The primer sequences used for real-time RT-PCR are designed using the Primer3 program and listed in S3 Table.