Laser capture microdissection and real-time reverse transcriptase/polymerase chain reaction of bronchiolar epithelium after bleomycin

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 bronch iolar epithelium. We used laser capture microdissection (LCM) to collect te rminal bronchiolar epithelial cells from frozen sections of lungs of mice s ubjected to intratracheal bleomycin. The RNA from these cells was used for analysis of select messenger RNAs (mRNAs) by quantitative real-time polymer ase chain reaction (PCR). In parallel, we used real-time PCR to analyze mRN As in whole-lung homogenates prepared from other mice given intratracheal b leomycin. We found reductions of Clara cell-specific protein and keratinocy te growth factor receptor mRNAs in both terminal bronchiolar epithelium and whole-lung homogenates 7 d after bleomycin. In contrast, terminal bronchio lar epithelial transforming growth factor (TGF)-alpha mRNA was reduced but whole-lung TGF-alpha mRNA was not changed, whereas terminal bronchiolar epi thelial epidermal growth factor (EGF) receptor mRNA was not changed but who le-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 te rminal bronchiolar epithelium is not necessarily reflected in analysis of w hole-lung gene expression.