Gene Expression Profiling and RNA visualization with Single-Molecule FISH

使用单分子 FISH 进行基因表达谱分析和 RNA 可视化

• 批准号: 8349532
• 负责人: Daniel Larson
• 金额: $ 25.61万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349532
• 关键词: Address; Algorithms; B-Cell Lymphomas; Bioinformatics; Biological; Biological Assay; Biological Process; Cataloging; Catalogs; Cell physiology; Cells; Chemicals; Classification; Communities; Computer software; Custom; DNA; DNA purification; Data; Data Set; Detection; Estrogens; Exhibits; Fluorescent in Situ Hybridization; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Genes; Goals; Housing; Image; Image Analysis; Imagery; Individual; Label; Laboratories; Libraries; Link; Measures; Messenger RNA; Meta-Analysis; Methodology; Methods; Microscope; Microscopic; Microscopy; Molecular Profiling; Monitor; National Cancer Institute; Noise; Oncogenes; Pathway interactions; Population; Process; Proteins; Publishing; RNA; Regulation; Research Personnel; Resolution; Role; Signal Transduction; Software Tools; Source; Spottings; Systems Biology; Testing; Time; Tissues; Variant; Work; base; chemical synthesis; design; fluorescence microscope; malignant breast neoplasm; programs; response; single molecule; transcriptomics; tumorigenesis

Single-molecule FISH on a large scale requires advances on multiple fronts. First, chemical synthesis, labeling and purification of DNA oligos must be accomplished. Second, hybridization and imaging in a 96-well plate format must be developed. Finally, automated image analysis and classification must be implemented. We have begun collaborating with Biosearch to provide labeled DNA oligos to the laboratory. In the past, there was no commercial source for FISH probes, and all synthesis was done on a small scale in-house. Thus, having a partner with significant synthesis capabilities removes a major obstacle to high-throughput FISH analysis. Since February 2011, when The Systems Biology of Gene Expression Section in the National Cancer Institute was established, a custom fluorescence microscope was designed, and the initial parts were purchased. This microscope will be optimized for single-molecule imaging and will also be capable of automated microscopy. In parallel, we have developed a new set of software tools for analyzing FISH data. These programs carry out spot-detection and segmentation along with cell segmentation. At present, we are testing the new algorithms on example data, but we anticipate this software will be made available to the scientific community. During the summer of 2011, Celine Hong was a summer intern in the section. During her time in the lab, she carried out a meta-analysis on two recently published data sets on breast cancer gene regulation. This work leads to direct predictions about the functional significance of co-regulation of genes during estrogen signaling. The testable hypotheses generated from this bioinformatic analysis will form the basis of several experimental efforts in the section in the next fiscal year. For example, we are designing a library of FISH probes to look at genes whose expression is linked to breast cancer. Some of the questions we will be addressing include: how frequently are these genes expressed in the population? Are genes involved in particular pathways co-expressed at the level of single cells? How much noise exists in the expression of these genes, and does this noise effect the process of tumorigenesis? When genes are up-regulated during breast cancer, are they uniformly up-regulated, or do certain cells exhibit strong responses while other cells exhibit weak responses?

单分子大规模的鱼需要在多个方面进步。首先，必须实现化学合成，标记和纯化。其次，必须开发出96孔板格式的杂交和成像。最后，必须实施自动图像分析和分类。我们已经开始与BioSearch合作，向实验室提供标记的DNA寡寡做。过去，没有鱼类探针的商业来源，所有合成都是在内部小规模完成的。因此，拥有具有重要合成能力的合作伙伴消除了高通量鱼类分析的主要障碍。自2011年2月以来，建立了国家癌症研究所的基因表达系统系统生物学，设计了定制的荧光显微镜，并购买了初始部分。该显微镜将针对单分子成像进行优化，并且还可以自动显微镜。同时，我们开发了一套新的软件工具来分析鱼类数据。这些程序以及细胞分割进行了点检测和分割。目前，我们正在测试示例数据的新算法，但是我们预计该软件将提供给科学界。 在2011年夏季，席琳·洪（Celine Hong）是该部分的暑期实习生。在实验室期间，她对最近发表的有关乳腺癌基因调节的数据集进行了荟萃分析。这项工作导致有关雌激素信号过程中基因共同调节功能意义的直接预测。 该生物信息学分析产生的可检验的假设将构成下一个财政年度本节中几项实验努力的基础。例如，我们正在设计一个鱼类探针库，以查看其表达与乳腺癌有关的基因。我们将要解决的一些问题包括：这些基因在人群中表达多久？在单个细胞水平上共表达的特定途径中是否涉及基因？这些基因的表达中存在多少噪声，这种噪声会影响肿瘤发生过程吗？当基因在乳腺癌期间上调时，它们是否均匀上调，或者某些细胞表现出强烈的反应，而其他细胞表现出较弱的反应？