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 BEC, AS+ and AS-. In AEC NOSII mRNA was not detectable.


CONCLUSIONS
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.
Department of Pathology, Justus Liebig University Giessen, Germany. Rainer.Bohle@patho.med.uni-giessen.de
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Bohle
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Hartmann
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Kinfe
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Ermert
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Seeger
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Fink
D000818:Animals; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D015925:Cryopreservation; D004210:Dissection / Q000295:instrumentation / Q000379:methods; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology; D005404:Fixatives; D005557:Formaldehyde; D011163:Hydroxymethylbilane Synthase / Q000096:biosynthesis / Q000235:genetics; D007834:Lasers; D008168:Lung / Q000737:chemistry* / Q000166:cytology* / Q000201:enzymology; D008297:Male; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D009928:Organ Specificity / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D016707:Tissue Fixation
D005404:Fixatives; D012333:RNA, Messenger; D005557:Formaldehyde; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D011163:Hydroxymethylbilane Synthase