6th ESACP Congress, Heidelberg, April 7-11, 1999

A020
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

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.