A high-throughput strategy for protein profiling in cell microarrays using automated image analysis
A high-throughput strategy for protein profiling in cell microarrays using automated image analysis
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DOI：
10.1002/pmic.200700199
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发表时间：
2007-06-01
期刊：
影响因子：
3.4
通讯作者：
Asplund, Anna
中科院分区：
文献类型：
--
作者：
Stromberg, Sara;Bjorklund, Marcus Gry;Asplund, Anna
抗体生产的进步使得免疫组织化学（IHC）亲和试剂的供应不断增加，组织微阵列（TMA）技术有助于同时分析多种组织中的蛋白质表达。然而，收集经验证的IHC数据仍然是一个瓶颈问题，因为标准方法是手动显微镜分析。在这里，我们提出了一种高通量的策略，结合免疫组化对最近开发的细胞微阵列与一种新的，自动化的图像分析应用程序（TMAx）。通过比较TMAx注释与由七位人类专家进行的手动注释，在200张IHC染色细胞斑点的数字图像上对该软件进行了评估。在一项有限的研究中，我们还研究了通过使用TMAx输出结果进行相对蛋白质定量和定量实时PCR进行相应转录水平定量来评估mRNA和蛋白质水平之间相关性的可能性。总之，在微阵列格式的体外培养的细胞中的免疫化学染色的自动化分析可用于高通量蛋白质分析，并且从相同细胞系提取RNA提供了在全球范围内比较转录和蛋白质表达的基础。
Advances in antibody production render a growing supply of affinity reagents for immunohistochemistry (IHC), and tissue microarray (TMA) technologies facilitate simultaneous analysis of protein expression in a multitude of tissues. However, collecting validated IHC data remains a bottleneck problem, as the standard method is manual microscopical analysis. Here we present a high-throughput strategy combining IHC on a recently developed cell microarray with a novel, automated image-analysis application (TMAx). The software was evaluated on 200 digital images of IHC-stained cell spots, by comparing TMAx annotation with manual annotation performed by seven human experts. A high concordance between automated and manual annotation of staining intensity and fraction of IHC-positive cells was found. in a limited study, we also investigated the possibility to assess the correlation between mRNA and protein levels, by using TMAx output results for relative protein quantification and quantitative real-time PCR for the quantification of corresponding transcript levels. In conclusion, automated analysis of immunohistochemically stained in vitro-cultured cells in a microarray format can be used for high-throughput protein profiling, and extraction of RNA from the same cell lines provides a basis for comparing transcription and protein expression on a global scale.