Real-time quantitative RT-PCR after laser-assisted cell picking.
The present study describes a technique for quantitation of mRNA in a few isotypic cells obtained from an intact organ structure by combining laser-assisted cell picking and real-time PCR. The microscopically controlled lasering of selected cells in stained tissue sections was applied to lung alveolar macrophages, which are unique in that they can alternatively be gathered as a pure cell population from intact lungs by bronchoalveolar lavage as a reference technique. TNF-alpha was chosen as the transcriptionally inducible target gene to be quantified in alveolar macrophages of control rat lung, as well as low- and high-challenge lungs stimulated by endotoxin and IFN-gamma nebulization. Online fluorescence detection for quantitation of the number of amplified copies was based on 5' nuclease activity of Taq polymerase cleaving a sequence-specific dual-labeled fluorogenic hybridization probe. A pseudogene-free sequence of PBGD served as an internal calibrator for comparative quantitation of target. A quick procedure and minimized loss of template were achieved by avoiding RNA extraction, DNase digestion and nested-PCR. Using this approach, we demonstrated dose-dependent manifold upregulation of the ratio of TNF-alpha mRNA copies per one copy of PBGD mRNA in alveolar macrophages of the challenged lungs. The quantitative data obtained from laser-picked alveolar macrophages were well matched with those of lavaged alveolar macrophages carried out in parallel. We suggest that this new combination of laser-assisted cell picking and real-time PCR has great promise for quantifying mRNA expression in a few single cells or oligocellular clusters in intact organs, allowing assessment of transcriptional regulation in defined cell populations.
Fink
L
L
Department of Pathology, Justus-Liebig-Universität Giessen, Germany.
Seeger
W
W
Ermert
L
L
Hänze
J
J
Stahl
U
U
Grimminger
F
F
Kummer
W
W
Bohle
R M
RM
D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology; D002469:Cell Separation / Q000379:methods*; D017931:DNA Primers; D018076:DNA, Complementary; D007371:Interferon-gamma / Q000494:pharmacology; D007834:Lasers; D008070:Lipopolysaccharides / Q000494:pharmacology; D008168:Lung / Q000187:drug effects / Q000276:immunology*; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D008070:Lipopolysaccharides; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D019914:Taq Polymerase