Accelerating Cancer Research with Single Cell Arrays

利用单细胞阵列加速癌症研究

基本信息

批准号：
7813696
负责人：
Heinz-Ulrich Guenter Weier
金额：
$ 11.8万
依托单位：
UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7813696
关键词：
Acetic Acids Address Affect Biological Assay Breast Cancer Patient Cancer cell line Cell Count Cell Culture Techniques Cell Cycle Stage Cell Nucleus Cells Chromatin Chromatin Fiber Classification Clinical Research Communities Cytogenetic Analysis Cytogenetics DNA DNA Probes DNA Sequence Rearrangement Detection Digestion Disease Excision Fine-needle biopsy Fluorescent in Situ Hybridization Formalin Freezing Funding Genomics Hand Harvest In Situ Individual Interphase Interphase Cell Karyotype Lesion Lymphocyte Malignant neoplasm of thyroid Mammalian Cell Metaphase Metaphase Spread Methanol Methods Modeling Noise Paraffin Embedding Patients Phenotype Premalignant Preparation Process Proliferating Proteins Protocols documentation RNA Recovery Research Residual state Resolution Sampling Signal Transduction Solid Specimen Stretching Supporting Cell Techniques Technology Testing Tissues Transcript United States National Institutes of Health Work anticancer research base cancer cell clinical practice crosslink design innovation interest neoplastic cell novel parent grant parent project response sample fixation technique development technology development tool tumor

Acetic Acids Address Affect Biological Assay Breast Cancer Patient Cancer cell line Cell Count Cell Culture Techniques Cell Cycle Stage Cell Nucleus Cells Chromatin Chromatin Fiber Classification Clinical Research Communities Cytogenetic Analysis Cytogenetics DNA DNA Probes DNA Sequence Rearrangement Detection Digestion Disease Excision Fine-needle biopsy Fluorescent in Situ Hybridization Formalin Freezing Funding Genomics Hand Harvest In Situ Individual Interphase Interphase Cell Karyotype Lesion Lymphocyte Malignant neoplasm of thyroid Mammalian Cell Metaphase Metaphase Spread Methanol Methods Modeling Noise Paraffin Embedding Patients Phenotype Premalignant Preparation Process Proliferating Proteins Protocols documentation RNA Recovery Research Residual state Resolution Sampling Signal Transduction Solid Specimen Stretching Supporting Cell Techniques Technology Testing Tissues Transcript United States National Institutes of Health Work anticancer research base cancer cell clinical practice crosslink design innovation interest neoplastic cell novel parent grant parent project response sample fixation technique development technology development tool tumor

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项目摘要

DESCRIPTION (provided by applicant): This application is submitted in response to NIH Notice Number NOT-OD-09-058 entitled 'NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications'. Current techniques for full karyotype analysis rely on metaphase spreads, and cells in interphase cannot be analyzed. Since cells obtained from tumors are rarely in metaphase, our ongoing research focuses on the development of technologies for the cytogenetic analysis of interphase cells. The parent grant entitled 'Accelerating cancer research with single cell arrays (SCAs)' addresses the sensitive detection of chromosomal changes such as small translocations or genomic imbalances in small numbers of cells isolated from breast or thyroid cancer cell lines. The parent project will demonstrate the feasibility that interphase cell nuclei can be extended reproducibly on a solid support and develop methods for the cytogenetic analysis of extended chromatin fibers. This Competing Revision Application will define the working limits of the SCA technology by analyzing a range of relevant samples that has been processed under conditions that are likely to produce partial DNA degradation. On the other hand, our preliminary studies have demonstrated that the analysis of specimens which have undergone minimal degradation is complicated by hybridization of probes to DNA transcripts, i.e., RNA present in these cells. Thus, a second objective of the proposed research is the development of techniques to increase signal-to-noise ratios through RNA digestion or blocking. The two Specific Aims of the present application are 1. Define the limits of the SCA technology by analyzing samples that have undergone DNA degradation. Most archival samples will have suffered from fixation and storage. To investigate how widely the SCA technology can be applied to samples of interest to the cancer research community, we will study the effects of DNA nicks or loss and protein cross-links in archival samples comprised of acetic acid: methanol fixed lymphocytes, frozen placental tissues as well formalin-fixed paraffin-embedded tissue blocks. 2. Increase signal-to-noise ratios and thus the detection sensitivity by removal of RNA from chromatin. Using extended chromatin preparations from breast and thyroid cancer cells harvested from mammalian cell cultures, we will develop protocols to enzymatically digest and remove RNAs that interfere with the assay or to block hybridization of DNA probes to residual RNA, while minimizing DNA loss. SCAs will become powerful tools in basic and applied/clinical research, where such a sensitive assay may support cell classifications, thereby benefiting patients with de novo translocations or premalignant lesions as well as cancer patients. Furthermore, SCAs will allow the analysis of very small samples regardless of their viability or cell cycle stage. This will open new avenues for the analysis of small samples like those obtained by fine needle biopsies as well as the analysis of circulating or exfoliated tumor cells.

描述（由申请人提供）：此申请是针对NIH通知号而提交的，而不是od-09-058，标题为“ NIH宣布恢复法案的可用性用于有竞争力的修订申请”。当前用于全核型分析的技术取决于中期扩散，并且无法分析相间的细胞。由于从肿瘤获得的细胞很少在中期中，因此我们正在进行的研究重点是开发用于间相细胞的细胞遗传学分析的技术。父母的授予标题为“用单细胞阵列加速癌症研究（SCAS）”，涉及染色体变化的敏感检测，例如较小的易位或从乳腺癌或甲状腺癌细胞系分离的少量细胞中的基因组失衡。父项目将证明可以在稳固支持下可重复扩展相间细胞核的可行性，并开发用于扩展染色质纤维的细胞遗传学分析的方法。该竞争性修订应用程序将通过分析在可能导致部分DNA降解的条件下处理的一系列相关样本来定义SCA技术的工作限制。另一方面，我们的初步研究表明，通过将探针与DNA转录本的杂交（即，这些细胞中存在的RNA）杂交，对经历最小降解的样品的分析变得复杂。因此，拟议的研究的第二个目标是开发通过RNA消化或阻断增加信噪比的技术。本应用程序的两个具体目的是1。通过分析经历DNA降解的样品来定义SCA技术的限制。大多数档案样本都将遭受固定和存储空间的困扰。为了研究如何将SCA技术应用于癌症研究界感兴趣的样品，我们将研究由乙酸组成的档案酸样品中的DNA入口或损失和蛋白质交联的效果：甲醇固定淋巴细胞，胎盘胎盘纤维细胞，Frozin frozin frozin frozin frozin frozin frozin-frozin-formanin膜膜膜膜 - 粘合剂 - 膜 - 膜培养基组织的组织块。 2。增加信噪比，从而通过从染色质中去除RNA来检测敏感性。使用从哺乳动物细胞培养物中收获的乳腺癌和甲状腺癌细胞的扩展染色质制剂，我们将开发方案以酶促消化并去除干扰测定或阻止DNA探针与残留RNA的杂交的RNA，同时最小化DNA损失。 SCAS将成为基本和应用/临床研究中的强大工具，在这种研究中，这种敏感的测定可能支持细胞分类，从而使从头易位或预易受先前病变的患者以及癌症患者受益。此外，SCA将允许对非常小的样品进行分析，而不管其生存力或细胞周期阶段如何。这将为分析小样品（例如通过细针活检获得的小样本）以及循环或去角质肿瘤细胞的分析提供新的途径。