Cell type-specific mRNA quantitation in non-neoplastic tissues after laser-assisted cell picking

Objective: Cell type-specific mRNA quantitation can be reliably performed after harvesting less than 20 cell profiles from haemalaun-stained cryosections by laser-assisted cell picking, Up to now it has been unclear to what extent these techniques can be used to analyze differential gene expression in complex tissues. Methods: Using a rat model of experimental endotoxin priming of the lung various pulmonary cell types were microdissected from isolated perfused and ventilated rat lungs after aerosol lipopolysaccharide/interferon-gamma stimulation. Results: Porphobilinogen deaminase housekeeping gene (PBGD) and nitric oxide synthase II (NOSII) mRNA in arterial endothelial cells (AEC), bronchiolar epithelial cells (BEC), alveolar septum containing monocytes/macrophages (AS+), alveolar septum without monocytes/macrophages (AS-) and intraluminar alveolar macrophages (AM) could be quantified by real-time RT-PCR, The strongest upregulation of NOSII mRNA occurred in AM, while minimal NOSII expression was detected in BEG, AS+ and AS-, In AEC NOSII mRNA was not detectable. Conclusion: The combination of laser microdissection and real-time RT-PCR is a valuable tool for the quantitative in situ characterization of differential gene expression within complex tissues. Copyright (C) 2001 S. Karger AG, Basel.