Am. J. Respir. Cell Mol. Biol., Volume 25, Number 3, September, 2001 278-284

Laser Capture Microdissection and Real-Time Reverse Transcriptase/ Polymerase Chain Reaction of Bronchiolar Epithelium after Bleomycin

Tomoko Betsuyaku, Gail L. Griffin, Mark A. Watson, and Robert M. Senior

Division of Pulmonary and Critical Care Medicine, Department of Medicine; and Laboratory Medicine, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri

Terminal airways are affected in many lung diseases and toxic inhalations. To elucidate the changes in terminal airways in these diverse situations it will be helpful to profile and quantify gene expression in terminal bronchiolar epithelium. We used laser capture microdissection (LCM) to collect terminal bronchiolar epithelial cells from frozen sections of lungs of mice subjected to intratracheal bleomycin. The RNA from these cells was used for analysis of select messenger RNAs (mRNAs) by quantitative real-time polymerase chain reaction (PCR). In parallel, we used real-time PCR to analyze mRNAs in whole-lung homogenates prepared from other mice given intratracheal bleomycin. We found reductions of Clara cell-specific protein and keratinocyte growth factor receptor mRNAs in both terminal bronchiolar epithelium and whole-lung homogenates 7 d after bleomycin. In contrast, terminal bronchiolar epithelial transforming growth factor (TGF)- mRNA was reduced but whole-lung TGF- mRNA was not changed, whereas terminal bronchiolar epithelial epidermal growth factor (EGF) receptor mRNA was not changed but whole-lung EGF receptor was reduced. We conclude that LCM can isolate terminal bronchiolar epithelial cells for studies of cell-specific gene expression by quantitative real-time PCR, and that cell-specific gene expression in terminal bronchiolar epithelium is not necessarily reflected in analysis of whole-lung gene expression.

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