RNA isolation and quantitative real-time PCR
Total RNA was extracted from frozen nasal tissues and hNECs using the mirVana miRNA Isolation Kit (Life Technologies, USA). Complementary DNA was synthesized in a 20 µl reaction volume from 1 µg total RNA using qScript™ cDNA SuperMix (Quanta BioDesign) according to the protocol of manufacturer. Relative genes expression was detected using SYBR green gene expression assays and was normalized to 2-ΔCTwith GAPDH and ribosomal protein L13a (RPL13A) as a housekeeping gene. The gene primer sequences used were shown inTable E2 .