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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A020
Cell-specific molecular analysis has become a major research focus in many
fields of biomedicine. UV laser assisted cell picking is one of the
procedures allowing reliable access to target cells from complex tissues and
does not interfere with RNA isolation and cDNA amplification. Using the
real time PCR (TaqMan-PCR, PE ABI Prism 7700 Sequence Detection System,
Foster City, CA, USA) after single cell microdissection (P.A.L.M. UV-laser
microbeam, Bernried, Germany) of alveolar macrophages(AM) - 10-15 cell
profiles each - we evaluated TNFa mRNA regulation in isolated, ventilated
and ex-vivo perfused lungs from Sprague-Dawley rats. AM were stimulated by
aerosolized lipopolysaccharide(LPS) and interferon g(IFNg). AM from
controls, low challenge and high challenge lungs were investigated. Rat
PBGD (porphobilinogen deaminase) housekeeping gene mRNA expression served as
the internal calibrator. 6h after LPS/IFNg stimulation significant
upregulation of TNFa mRNA was detected by real time quantification. The
relative TNFa mRNA expression detected in selectively harvested AM from
hematoxylin stained cryosections amounted to 0.41+/-0.10 in control lungs,
2.07+/-0.39 in low challenge lungs as compared to 16.42+/-4.92 in
high-challenge lungs. Thus, a stimulation dependent upregulation of the
TNFa mRNA expression can be demonstrated in rat AM.
We suggest that laser assisted cell picking and mRNA quantification by
real time PCR has great promise to analyse mRNA regulation in a few single
cell profiles from complex tissues.
REAL-TIME QUANTIFICATION OF GENE EXPRESSION AFTER LASER- ASSISTED CELL
PICKING
Bohle RM 1, Seeger W 2, Ermert L 1, Haenze J 3, Stahl U 1,
Grimminger F 2, Kummer W 4, Fink L 1,2
1) Department of Pathology, 2) Internal Medicine, 3) Pediatrics and
4) Institute for Anatomy and Cell Biology, Justus-Liebig-University
Giessen, Germany