title: Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro.
abstract: Polarized and pseudostratified primary airway epithelia present barriers that significantly reduce their transfection efficiency and the efficacy of RNA interference oligonucleotides. This creates an impediment in studies of the airway epithelium, diminishing the utility of loss-of-function as a research tool. Here we outline methods to introduce RNAi oligonucleotides into primary human and porcine airway epithelia grown at an air-liquid interface and difficult-to-transfect transformed epithelial cell lines grown on plastic. At the time of plating, we reverse transfect small-interfering RNA (siRNA), Dicer-substrate siRNA, or microRNA oligonucleotides into cells by use of lipid or peptide transfection reagents. Using this approach we achieve significant knockdown in vitro of hypoxanthine-guanine phosphoribosyltransferase, IL-8, and CFTR expression at the mRNA and protein levels in 1-3 days. We also attain significant reduction of secreted IL-8 in polarized primary pig airway epithelia 3 days posttransfection and inhibition of CFTR-mediated Cl⁻ conductance in polarized air-liquid interface cultures of human airway epithelia 2 wk posttransfection. These results highlight an efficient means to deliver RNA interference reagents to airway epithelial cells and achieve significant knockdown of target gene expression and function. The ability to reliably conduct loss-of-function assays in polarized primary airway epithelia offers benefits to research in studies of epithelial cell homeostasis, candidate gene function, gene-based therapeutics, microRNA biology, and targeting the replication of respiratory viruses.
authors.affiliation: Department of Pediatrics, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Shyam
authors.initials: S
authors.lastname: Ramachandran
authors.forename: Sateesh
authors.initials: S
authors.lastname: Krishnamurthy
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Jacobi
authors.forename: Christine
authors.initials: C
authors.lastname: Wohlford-Lenane
authors.forename: Mark A
authors.initials: MA
authors.lastname: Behlke
authors.forename: Beverly L
authors.initials: BL
authors.lastname: Davidson
authors.forename: Paul B
authors.initials: PB
authors.lastname: McCray
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics / Q000473:pathology; D000818:Animals; D015153:Blotting, Western; D016764:Cell Polarity; D002478:Cells, Cultured; D003110:Colonic Neoplasms / Q000235:genetics / Q000473:pathology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004594:Electrophysiology; D004847:Epithelial Cells / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D007124:Immunoenzyme Techniques; D066298:In Vitro Techniques; D016209:Interleukin-8 / Q000378:metabolism; D008297:Male; D009841:Oligonucleotides / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020545:Respiratory Mucosa / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine
chemical_list: D016209:Interleukin-8; D009841:Oligonucleotides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: RNA interference; air-liquid interface; gene silencing; porcine airway epithelial cells; primary airway epithelium; siRNA; transfection