Trizol reagent

Reverse transcription was completed using the SPARKscript II RT Plus Kit (Sparkjade, Jinan, China), after total RNA was extracted using Trizol reagent (Sparkjade, China). Using 2 × SYBR Green qPCR Mix (With ROX) (Sparkjade, China), real-time PCR was performed on a LightCycler 480 real-time PCR machine (Roche, Basel, Switzerland). The exact primers (Azenta, Suzhou, China) were utilized to amplify the genes for Collagen I (Coli), alkaline phosphatase (Alp), runt-related transcription factor 2 (Runx2), and glyceraldehyde-3-phosphate dehydrogenase (Gapdh). Cycle threshold (Ct) values were utilized to compare relative mRNA expression, while Gapdh was used as an internal control. The experimental data were computed utilizing the 2^−ΔΔCt approach [46 (link)]. Each experiment was repeated three times.

Total RNA was extracted with Trizol reagent (Sparkjade AC010, Jinan, China) and concentration of sample RNA was detected by a spectrophotometer. The reverse transcription kit (Sparkjade AG0304, Jinan, China) was used to reverse 1 μg RNA to cDNA. The real-time fluorescence quantitative PCR telomerase detection kit (KeyGen BioTech KGA1028R) was used to configure the qPCR mix 25 μL system according to the instructions. TRET and GAPDH genes were amplified by two steps: 95°C for 10 min, predenatured, 95°C for 15 s, annealed at 60°C and extended for 1 min, and amplified for 40 cycles. The relative expression of telomerase activity mRNA in each group was calculated according to the CT value. The primer sequences were as follows:
TRET: forward primer 5′-GACATGGAGAACAAGCTGTTTGC-3′; reverse primer 5′-ACAGGGAAGTTCACCACTGTC-3′; GAPDH: forward primer 5′-AGGTTGTCTCCTGTGACTTCAA-3′; reverse primer 5′-CTGTTGCTGTAGCCATATTCATTG-3′.

Total RNAs were isolated from cells using TRIzol reagent (SparkJade, Qingdao, China) according to the manufacturer's instructions. The concentration and purity of total RNAs were determined spectrophotometrically by measuring the absorbance at 260 nm and 280 nm using a UV spectrophotometer, and cDNA was produced using an ABI Veriti 96‐Well Thermal Cycler (Waltham, MA, USA) and FastQuant RT Kit with gDNase (Tiangen). Real‐time PCR was performed in an ABI QuantStudio3 PCR System (Waltham, MA, USA) using SYBR Green qPCR Master Mix and gene‐specific primers with an initial denaturation step at 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds, 58°C for 30 seconds and 68°C for 60 seconds. The primers for qRT‐PCR are listed in Table 1. The fold change in the expression of targets relative to the housekeeping gene GAPDH was calculated based on the 2^‐ΔΔCt relative expression formula.

Total RNA was isolated from the tissue samples using Trizol reagent following manufacturer’s instructions (Sparkjade, Qingdao, China). Then 1 μg RNA of each sample was reverse-transcribed to obtain cDNA using SPARKscript II RT Plus Kit (With gDNA Eraser) (Sparkjade, Qingdao, China). The expression of circRNAs and mRNAs in these individual samples was performed by qRT-PCR reaction using SYBR Green qPCR Mix kit (With ROX) (Sparkjade, Qingdao, China) following: 94° C for 2 min, followed by 40 cycles of 95° C for 10 s and 60° C for 30 s. The extraction and amplification of miRNAs was performed according to the manufacturer’s instructions (SPARKeasy animal tissue/Cell microRNA kit, miRNA first strand synthesis kit and miRNA SYBR Green qPCR Mix kit, Sparkjade, Qingdao, China). All RT-qPCR were repeated three times. The relative expression of all genes was calculated using the 2^-ΔΔCT method.

Total RNA was extracted from mouse nasal mucosa and human peripheral blood mononuclear cells (PBMCs) using the Trizol reagent (SparkJade, Qingdao, China) and reverse transcribed into cDNA using a reverse transcription kit (Accurate Bio, Changsha, China). The qPCR step was performed using an SYBR Green qPCR Mix kit (SparkJade) in a StepOnePlus fluorescence quantitative PCR instrument (Applied Biosystems, Foster City, CA, United States). The expression levels of the genes encoding IL-4, IL-5, epidermal growth factor receptor (EGFR), mitogen-activated protein kinase 1 (MAPK1), and protein kinase B1 (AKT1) were detected. The relative expression level was calculated using the 2^–ΔΔCT method (Livak and Schmittgen, 2001 (link)). The primers for mouse Egfr, Mapk1, and Akt1 and human IL4, IL5, EGFR, MAPK1, and AKT1 are listed in Supplementary Table 1.