Journal article
Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro
American journal of physiology. Lung cellular and molecular physiology, Vol.305(1), pp.L23-L32
07/01/2013
DOI: 10.1152/ajplung.00426.2012
PMCID: PMC4073929
PMID: 23624792
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.
Details
- Title: Subtitle
- Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro
- Creators
- Shyam Ramachandran - Department of Pediatrics, University of Iowa, Iowa City, IowaSateesh Krishnamurthy - Department of Internal Medicine, University of Iowa, Iowa City, IowaAshley M Jacobi - Integrated DNA Technologies, Coralville, IowaChristine Wohlford-Lenane - Department of Pediatrics, University of Iowa, Iowa City, IowaMark A Behlke - Integrated DNA Technologies, Coralville, IowaBeverly L Davidson - Department of Internal Medicine, University of Iowa, Iowa City, IowaPaul B McCray - Department of Pediatrics, University of Iowa, Iowa City, Iowa
- Resource Type
- Journal article
- Publication Details
- American journal of physiology. Lung cellular and molecular physiology, Vol.305(1), pp.L23-L32
- DOI
- 10.1152/ajplung.00426.2012
- PMID
- 23624792
- PMCID
- PMC4073929
- NLM abbreviation
- Am J Physiol Lung Cell Mol Physiol
- ISSN
- 1040-0605
- eISSN
- 1522-1504
- Publisher
- American Physiological Society
- Language
- English
- Date published
- 07/01/2013
- Academic Unit
- Microbiology and Immunology; Pulmonary Medicine; Stead Family Department of Pediatrics; Internal Medicine
- Record Identifier
- 9984093217702771
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