Journal
EUROPEAN JOURNAL OF CELL BIOLOGY
Volume 101, Issue 1, Pages -Publisher
ELSEVIER GMBH
DOI: 10.1016/j.ejcb.2021.151189
Keywords
Airway tissue differentiation; genetic disorders; ciliogenesis; Gene expression; TLDA; qRT-PCR; Bulk RNAseq; Protein expression; Immunofluorescence; Primary ciliary dyskinesia genes
Categories
Funding
- National Science Centre (Poland) [DEC-2013/09/D/NZ4/01692, DEC-2018/31/B/NZ2/03248]
Ask authors/readers for more resources
The study utilized TLDA and qRT-PCR methods to analyze the expression of 14 genes in human airway epithelial cells, showing that cilia-related genes were upregulated over culture time and visualized through immunofluorescent staining.
Primary cultures of the human airway epithelium (AE) cells are an indispensable tool in studies of pathophysiology of genetic and environmental pulmonary diseases, including cystic fibrosis (CF), primary ciliary dyskinesia (PCD) and chronic obstructive pulmonary disease (COPD). Air-liquid interface (ALI) culture is the best method to follow the differentiation of ciliated cells, whose dysfunction forms the basis of PCD. Here, we used customdesigned Taqman Low Density Array (TLDA), qRT-PCR-based assay, to analyze expression of 14 AE genes in cells from healthy donors, cultured in ALI settings using Pneumacult medium, with the focus on genes involved in cilia differentiation and in PCD pathogenesis. The results of TLDA assay were compared with the bulk RNAseq analysis, and placed in the cellular context using immunofluorescent staining (IF) of ALI cultured cells. Expression analysis revealed culture time-related upregulation of the majority of cilia-related genes, followed by the appearance of respective protein signals visualized by IF. Strong correlation of TLDA with RNAseq results indicated that TLDA assay is a reliable and scalable approach to analyze expression of selected genes specific for different AE cell types. Characterization of temporal and inter-donor changes in the expression of these genes, performed in healthy donors and in well-defined ALI/Pnemacult culture conditions, provides a useful reference relevant for a broad spectrum of functional studies where the in vitro AE differentiation is in focus.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available