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 .