Macdonald Jennifer A, Murugesan Nivetha, Pachter Joel S
Blood-Brain Barrier Laboratory, Center for Vascular Biology and Department of Cell Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3505, USA.
J Neurosci Methods. 2008 Sep 30;174(2):219-26. doi: 10.1016/j.jneumeth.2008.07.009. Epub 2008 Jul 23.
Laser capture microdissection (LCM) holds great potential for analyzing gene expression profiles in situ. Most recently, this laboratory employed a novel immunostain-based LCM protocol (immuno-LCM) to selectively retrieve brain microvascular endothelial cells (BMEC) from intimately associated perivascular cells. However, before this protocol can be confidently coupled to downstream analytical platforms, it must be demonstrated that any variability associated with it is minimal, so as not to obscure data interpretation. As various factors could contribute to variability, this study focused on determining whether technical inconsistency and/or biological diversity of sample populations, played such a role. Specifically, two separate immuno-LCM-derived BMEC samples derived from adjacent tissue sections of a single mouse (to detect only technical variability), and from analogous tissue sections of three different mice (to detect technical and biological variability) were compared for their relative expression of 16 genes, using quantitative-RT-PCR (qRT-PCR). Both significant linear and rank-order correlations were observed between different sections from the same animal, underscoring lack of technical variability in this LCM application. Furthermore, a three-dimensional scatter plot of gene expression profiles from the three animals was linear, and ANOVA showed absence of statistically significant differences between any of the animals, confirming lack of biological variability. These findings argue that immuno-LCM coupled to qRT-PCR affords a reproducible means to assay gene expression in situ.
激光捕获显微切割（LCM）在原位分析基因表达谱方面具有巨大潜力。最近，本实验室采用了一种基于新型免疫染色的LCM方案（免疫LCM），从紧密相连的血管周围细胞中选择性地获取脑微血管内皮细胞（BMEC）。然而，在该方案能够可靠地与下游分析平台结合之前，必须证明与之相关的任何变异性都是最小的，以免模糊数据解读。由于各种因素可能导致变异性，本研究着重确定样本群体的技术不一致性和/或生物多样性是否起了这样的作用。具体而言，使用定量逆转录聚合酶链反应（qRT-PCR）比较了来自同一只小鼠相邻组织切片的两个独立的免疫LCM衍生的BMEC样本（仅检测技术变异性）以及来自三只不同小鼠类似组织切片的样本（检测技术和生物变异性）中16个基因的相对表达。在同一动物的不同切片之间观察到了显著的线性和秩次相关性，强调了该LCM应用中缺乏技术变异性。此外，来自三只动物的基因表达谱的三维散点图呈线性，方差分析表明任何一只动物之间均无统计学上的显著差异，证实了缺乏生物变异性。这些发现表明，免疫LCM与qRT-PCR相结合为原位检测基因表达提供了一种可重复的方法。
