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Real-time quantitative RT-PCR after laser-assisted cell picking.

Abstract：

: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.

journal_name

Nat Med

journal_title

Nature medicine

authors

Fink L,Seeger W,Ermert L,Hänze J,Stahl U,Grimminger F,Kummer W,Bohle RM

doi

10.1038/3327

subject

Has Abstract

pub_date

1998-11-01 00:00:00

pages

1329-33

issue

11

eissn

1078-8956

issn

1546-170X

journal_volume

4

pub_type

杂志文章

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