A Quantitative Comparison of Cell-Type-Specific Microarray Gene Expression Profiling Methods in the Mouse Brain

Expression profiling of restricted neural populations using microarrays can facilitate neuronal classification and provide insight into the molecular bases of cellular phenotypes. Due to the formidable heterogeneity of intermixed cell types that make up the brain, isolating cell types prior to microarray processing poses steep technical challenges that have been met in various ways. These methodological differences have the potential to distort cell-type-specific gene expression profiles insofar as they may insufficiently filter out contaminating mRNAs or induce aberrant cellular responses not normally present in vivo. Thus we have compared the repeatability, susceptibility to contamination from off-target cell-types, and evidence for stress-responsive gene expression of five different purification methods - Laser Capture Microdissection (LCM), Translating Ribosome Affinity Purification (TRAP), Immunopanning (PAN), Fluorescence Activated Cell Sorting (FACS), and manual sorting of fluorescently labeled cells (Manual). We found that all methods obtained comparably high levels of repeatability, however, data from LCM and TRAP showed significantly higher levels of contamination than the other methods. While PAN samples showed higher activation of apoptosis-related, stress-related and immediate early genes, samples from FACS and Manual studies, which also require dissociated cells, did not. Given that TRAP targets actively translated mRNAs, whereas other methods target all transcribed mRNAs, observed differences may also reflect translational regulation.利用微阵列（microarray）对特定神经亚群开展表达谱分析，可有效助力神经元分类，并为解析细胞表型的分子基础提供重要视角。由于构成大脑的混合细胞类型具有极强的异质性，在微阵列分析前分离目标细胞类型面临着极高的技术挑战，目前已有多种技术策略得以解决此类难题。此类不同的实验方法可能会扭曲细胞类型特异性基因表达谱，究其原因在于：它们或无法充分过滤污染的信使RNA（mRNA），或会诱导出体内正常状态下不会出现的异常细胞应答。为此，我们对比了五种不同纯化方法的重复性、脱靶细胞类型污染易感性，以及应激响应基因表达的相关证据，这五种方法分别为：激光捕获显微切割（Laser Capture Microdissection, LCM）、翻译核糖体亲和纯化（Translating Ribosome Affinity Purification, TRAP）、免疫淘选（Immunopanning, PAN）、荧光激活细胞分选（Fluorescence Activated Cell Sorting, FACS），以及荧光标记细胞手动分选（Manual）。研究结果显示，所有方法均实现了相当高的重复性水平；但激光捕获显微切割与翻译核糖体亲和纯化所得数据的污染程度，显著高于其余三种方法。免疫淘选样本的凋亡相关、应激相关以及即刻早期基因的激活水平更高，而同样需要对细胞进行解离处理的荧光激活细胞分选与手动分选样本则未出现此类激活现象。鉴于翻译核糖体亲和纯化靶向的是活跃翻译的mRNA，而其余方法靶向的是所有已转录的mRNA，本次观测到的实验差异也可能反映了翻译调控过程。