Rotor gene rg 300

To examine adeB gene expression, isolated were treated with MIC and sub MIC of CCCP. RNA from each isolate was extracted using RNX- Plus reagent (SinaClon BioScience; Iran) solution according to the manufacturer’s instructions. The Thermo Scientific^™ NanoDrop 2000 calculated the concentration and purification of each RNA sample. cDNAs were synthesized using the Revert Aid first-strand cDNA synthesis kit (Yektatajhiz; Iran) with 1.5 μg of RNA at a 20 μL reaction rate. The Real-Time PCR was conducted on a Rotor-Gene RG-300 (Corbett Research, Sydney, AU) and SYBR Green Real-time PCR Master Mix kit (Yektatajhiz; Iran) to show the quantification of mRNA. A paired primer was applied for the amplification of adeB gene (primer sequence: F: 5'-AACGGACGACCATCTTTGAG-3'; R: 5'-CAGTTGTTCCATTTCACGCA-3'). Thermal cycling continued the denaturation process at 95 °C for 5 min for the first denaturation stage at 95 °C for 5 min and then 38 cycles at 95 °C for 15 s, 61 °C for 20 and 72 for 25 s. The amplification specificity of adeB gene expression was confirmed by the melting curve. adeB gene expression was standardized against 16sRNA as an internal control, and relative quantification (2^−ΔΔCq) revealed the fold changes of expression.

Total RNA was extracted from the nasal tissue samples using Biozol solution (Bioflux; Japan). At 260/280 and 260/230 wavelength absorption ratios, the concentration of RNA was calculated by Nanodrop™ 2000c (Thermo Fisher Scientific, IL, USA). To cut down on any DNA contamination, 10 u/ml DNaseI were added to 1 mg/ml RNA samples for 30 min. Then, the Revert Aid firststrand cDNA synthesis kit (Thermo Scientific; Lithuania) was used to synthesize cDNA from total RNA by incubation at 25°C for 5 min, at 42°C for 1 h, and for 5 min at 72°C in a thermocycler. SYBR Green master mix (Takara; Japan) was used for the real-time PCR, which was performed in 35 cycles at a Rotor-Gene RG-300 (Corbett Research, Sydney, AU). Briefly, denaturation was carried out at 95°C for 10 min followed by incubation at 95°C for 15 s, 57°C for 20 s, and 72°C for 25 s. The specific primers listed in Table 2 were used to evaluate the expression of the TGF-β1, IL-6, IL-18, IL-21, and IL-23. β-actin gene was used to normalize the data. The standard curves were drawn to evaluate the primers' efficiency, which was above 90% on average. The data were expressed as fold change using the comparative cycle threshold (2-ΔΔct method).