Fink L, Seeger W, Ermert L, Hänze J, Stahl U, Grimminger F, Kummer W, Bohle R M
Department of Pathology, Justus-Liebig-Universität Giessen, Germany.
Nat Med. 1998 Nov;4(11):1329-33. doi: 10.1038/3327.
The present study describes a technique for quantitation of mRNA in a few isotypic cells obtained from an intact organ structure by combining laser-assisted cell picking and real-time PCR. The microscopically controlled lasering of selected cells in stained tissue sections was applied to lung alveolar macrophages, which are unique in that they can alternatively be gathered as a pure cell population from intact lungs by bronchoalveolar lavage as a reference technique. TNF-alpha was chosen as the transcriptionally inducible target gene to be quantified in alveolar macrophages of control rat lung, as well as low- and high-challenge lungs stimulated by endotoxin and IFN-gamma nebulization. Online fluorescence detection for quantitation of the number of amplified copies was based on 5' nuclease activity of Taq polymerase cleaving a sequence-specific dual-labeled fluorogenic hybridization probe. A pseudogene-free sequence of PBGD served as an internal calibrator for comparative quantitation of target. A quick procedure and minimized loss of template were achieved by avoiding RNA extraction, DNase digestion and nested-PCR. Using this approach, we demonstrated dose-dependent manifold upregulation of the ratio of TNF-alpha mRNA copies per one copy of PBGD mRNA in alveolar macrophages of the challenged lungs. The quantitative data obtained from laser-picked alveolar macrophages were well matched with those of lavaged alveolar macrophages carried out in parallel. We suggest that this new combination of laser-assisted cell picking and real-time PCR has great promise for quantifying mRNA expression in a few single cells or oligocellular clusters in intact organs, allowing assessment of transcriptional regulation in defined cell populations.
本研究描述了一种通过结合激光辅助细胞挑选和实时PCR技术，对从完整器官结构中获取的少量同型细胞中的mRNA进行定量分析的方法。在染色组织切片中对选定细胞进行显微镜控制的激光照射，应用于肺泡巨噬细胞，其独特之处在于，作为一种参考技术，它们也可以通过支气管肺泡灌洗从完整的肺中收集为纯细胞群体。选择肿瘤坏死因子-α（TNF-α）作为转录诱导靶基因，在对照大鼠肺、以及由内毒素和干扰素-γ雾化刺激的低剂量和高剂量攻击的肺的肺泡巨噬细胞中进行定量分析。基于Taq聚合酶切割序列特异性双标记荧光杂交探针的5'核酸酶活性，进行在线荧光检测以定量扩增拷贝数。PBGD的无假基因序列用作靶标比较定量的内部校准物。通过避免RNA提取、DNA酶消化和巢式PCR，实现了快速程序和模板损失最小化。使用这种方法，我们证明了在受攻击肺的肺泡巨噬细胞中，每一个PBGD mRNA拷贝的TNF-α mRNA拷贝数的比例呈剂量依赖性的多倍上调。从激光挑选的肺泡巨噬细胞获得的定量数据与同时进行的灌洗肺泡巨噬细胞的数据非常匹配。我们认为，激光辅助细胞挑选和实时PCR的这种新组合，对于定量完整器官中少数单细胞或寡细胞簇中的mRNA表达具有很大的前景，能够评估特定细胞群体中的转录调控。
