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Highly multiplexed ion-beam tissue RNA in situ imaging with sub-micron resolution

亚微米分辨率的高度多重离子束组织 RNA 原位成像

基本信息

批准号：
8928079
负责人：
ALEXANDER D BOROWSKY
金额：
$ 26.29万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-16 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8928079
关键词：
Amplifiers Antibodies Binding Biological Biological Assay Biology Cancer Biology Carcinoma in Situ Cell Line Cell division Cells Cellular biology Clinical Code Complex DNA DNA Sequence Data Detection Development Dimensions Evaluation Flow Cytometry Formalin Gene Expression Generations Genes Genetic Transcription Health Histology Human Image Immune system In Situ In Situ Hybridization Individual Ions Label Laboratories Lanthanoid Series Elements Laser Scanning Confocal Microscopy Lasers Light Location Malignant Neoplasms Messenger RNA Metals Methodology Methods Microscopic Minor Molecular Molecular Profiling Monitor Mus Neoplasm Metastasis Noise Outcome Oxygen Paraffin Embedding Performance Phenotype Play Preparation RNA RNA Sequences Reporter Reproducibility Research Resolution Role Sampling Scanning Science Sensitivity and Specificity Shapes Signal Pathway Signal Transduction Solutions Specimen Techniques Technology Tissues Transcript Tumor Tissue Untranslated RNA Weight Work base cDNA Probes cancer therapy clinically relevant detector fluorophore imaging modality insight malignant breast neoplasm next generation next generation sequencing protein complex single molecule submicron tool transcriptome sequencing translational medicine tumor

项目摘要

DESCRIPTION (provided by applicant): Messenger and noncoding RNAs expression play a central role in shaping tumor development and clinical cancer outcomes. However, it is not possible to fully appreciate their functions in the absence of spatial context information. This application seeks to develop mass-tag-based molecularly specific imaging methods that can will be able to detect as many as 40 to 100+ in situ hybridization (ISH) RNA targets in morphologically intact cells and tissues, simultaneously and with single-molecule sensitivity. The technique is analogous to laser-scanning confocal microscopy, except that the scanning is performed using a tightly focused ion beam, rather than a similarly sized point of laser light. And instead of fluorophores, the labels detected are clusters of metal atoms attached to individual probes. When ionized and collected at a detector, these metal species are unambiguously distinguished based on their mass (atomic weight). Preliminary data using a highly focused, scanning oxygen ion beam and 10 antibodies applied simultaneously to clinical formalin-fixed, paraffin- embedded breast cancer tissues demonstrate histology-like images that reveal bound antibody location with subcellular resolution. We will extend this work to develop and validate similar methods for the simultaneous detection of multiple different coding and/or noncoding RNA species, particularly those with proven or suspected significance for cancer biology and translational medicine. The RNA labeling technology will be based on the related but non-imaging approach of mass-tag-based flow cytometry developed in the Nolan laboratory. This labeling strategy relies in part on paired initial cDNA probes that must bind to adjacent sequences to generate a secondary target for branched-chain metal-labeled DNA reporters. The initial double- binding requirement dramatically decreases the impact of non-specific binding on ultimate signal generation. First steps will be to demonstrate the feasibility of mass-tagged multiplexed detection of RNA species. We will optimize labeling and binding parameters necessary to achieve at least 10X multiplexing, and will determine achievable sensitivity and specificity. We will then explore effects of pre-analytical variables including sample preparation methodologies. Finally, to document potential utility, we will examine cell lines with well- documented quantitative RNA expression profiles, as well as mouse and human clinical breast cancer specimens using relevant probe targets. Implications: The methods outlined here will allow spatially resolved evaluation of numerous signaling pathways simultaneously, and will also shed light on interactions between cells whose detailed individual phenotypes we can assess. In addition, at the subcellular level, we anticipate that the ability to detect, in three- dimensions, multiple RNAs and RNA-protein complexes with ~50-nm isotropic resolution will open up new vistas in basic and translational cell biology. Clinically, we expect to adapt existing gene-expression panel analysis to intact tissues-with an ability to monitor relevant but possibly minor tumor subpopulations.

描述（由申请人提供）：信使和非编码RNA表达在形成肿瘤发展和临床癌症结局中发挥核心作用。然而，在缺乏空间背景信息的情况下，不可能完全理解它们的功能。该申请旨在开发基于质量标签的分子特异性成像方法，该方法能够同时并以单分子灵敏度检测形态完整的细胞和组织中多达40至100+个原位杂交（ISH）RNA靶标。该技术类似于激光扫描共聚焦显微镜，除了使用紧密聚焦的离子束进行扫描，而不是类似大小的激光点。和检测的标记不是荧光团，而是附着在单个探针上的金属原子簇。当离子化并在检测器处收集时，这些金属物质基于它们的质量（原子量）被明确区分。使用高度聚焦的扫描氧离子束和同时应用于临床福尔马林固定的石蜡包埋的乳腺癌组织的10种抗体的初步数据证明了以亚细胞分辨率揭示结合抗体位置的组织学样图像。我们将扩展这项工作，以开发和验证类似的方法，用于同时检测多个不同的编码和/或非编码RNA种类，特别是那些已证实或怀疑对癌症生物学和转化医学具有重要意义的RNA种类。RNA标记技术将基于Nolan实验室开发的基于质量标签的流式细胞术的相关但非成像方法。这种标记策略部分依赖于成对的初始cDNA探针，这些探针必须与邻近序列结合，以产生支链金属标记的DNA报告基因的二级靶标。初始的双结合要求显著降低了非特异性结合对最终信号产生的影响。第一步将是证明RNA种类的质量标记多重检测的可行性。我们将优化实现至少10倍多重化所需的标记和结合参数，并将确定可实现的灵敏度和特异性。然后，我们将探讨分析前变量的影响，包括样品制备方法。最后，为了记录潜在的效用，我们将使用相关的探针靶标检查具有充分记录的定量RNA表达谱的细胞系，以及小鼠和人临床乳腺癌标本。含义：这里概述的方法将允许同时对许多信号通路进行空间分辨评估，并且还将揭示我们可以评估其详细的个体表型的细胞之间的相互作用。此外，在亚细胞水平上，我们预计在三维空间中检测的能力，具有~50 nm各向同性分辨率的多个RNA和RNA-蛋白质复合物将在基础和翻译细胞生物学中开辟新的前景。在临床上，我们期望将现有的基因表达分析应用于完整的组织，并有能力监测相关但可能较小的肿瘤亚群。