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 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)-alpha mRNA was reduced but whole-lung TGF-alpha 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.
Betsuyaku
T
T
Division of Pulmonary and Critical Care Medicine, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Griffin
G L
GL
Watson
M A
MA
Senior
R M
RM
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001761:Bleomycin / Q000494:pharmacology*; D001980:Bronchi / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D016591:Histocytological Preparation Techniques; D007834:Lasers; D051379:Mice; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D051497:Receptor, Fibroblast Growth Factor, Type 2; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics / Q000378:metabolism; D020545:Respiratory Mucosa / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013223:Statistics as Topic; D016211:Transforming Growth Factor alpha / Q000235:genetics / Q000378:metabolism; D014598:Uteroglobin
D000903:Antibiotics, Antineoplastic; D004791:Enzyme Inhibitors; D011506:Proteins; D017468:Receptors, Fibroblast Growth Factor; C556390:Scgb1a1 protein, mouse; D016211:Transforming Growth Factor alpha; D001761:Bleomycin; D014598:Uteroglobin; D066246:ErbB Receptors; D051497:Receptor, Fibroblast Growth Factor, Type 2; C065104:keratinocyte growth factor receptor