nCounter Prep Station and the Digital Analyzer

nCounter Prep Station 和数字分析仪

• 批准号: 7793214
• 负责人: Laurence J.N. Cooper
• 金额: $ 23.58万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-26 至 2011-01-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7793214
• 关键词: Active immunity; Animals; Applications Grants; Biological Assay; Biological Response Modifier Therapy; Cancer Immunology Science; Cells; Clinical; Detection; Enzymes; Flow Cytometry; Formalin; Gene Expression; Gene Expression Profile; Genes; Homing; Human; Image; Immune; Immunophenotyping; Immunotherapeutic agent; Immunotherapy; Infusion procedures; Institution; Malignant Neoplasms; Measurement; Messenger RNA; Modern Medicine; Molecular; Molecular Profiling; Paraffin Embedding; Patients; Phenotype; RNA; Reaction; Research; Research Personnel; Role; Sampling; Serological; Specificity; System; Systems Analysis; Technology; Testing; Therapeutic; Tissue Sample; Tissues; Transcript; University of Texas M D Anderson Cancer Center; Whole Blood; Work; base; conventional therapy; design; digital; improved; mRNA Expression; new technology; next generation; novel; programs; protein expression; single molecule; tissue/cell culture; tumor

DESCRIPTION (provided by applicant): Cell-based immunotherapies hold great promise for treatment of malignancies, yet they are among the greatest challenges to develop and implement in modern medicine. There are typically no or few markers such as distinct clinical features, specific morphological changes by imaging, or serological identifiers, which can predict efficacy or even therapeutic potential of these biologic therapies. Despite these hurdles, and the challenge of generating cell-based immunotherapies from within not-for-profit academic institutions, immune- based therapies are being developed and tested in humans based on the premise and promise of their specificity and ability to eradicate tumor using effector mechanisms that are non-overlapping with conventional therapies. The Center for Cancer Immunology Research (CCIR) at University of Texas (UT) M.D. Anderson Cancer Center (MDACC) has an active program in designing and implementing clinical-grade immunotherapies based on enhancing passive and active immunity. Currently, the phenotype of cells infused and cells recovered from patients (and animals) are characterized by flow cytometry for protein expression and a limited analysis of expressed genes. However, it is vital to be able to classify immune genes that are differentially expressed before and after immunotherapy to gauge the therapeutic potential of the treatment and to undertake the rational design of next generation immunotherapeutics with predicted improved biologic effect. The two current approaches to profiling immune gene expression, microanalysis and Q-PCR, have a limited role in enhancing the potential of immunotherapy due to the large amount of material needed, with the resultant bias that occurs when mRNA is limiting and does not cover the array's dynamic concentration range, or the bias occurring with enzymatic amplification. Simply put, comprehensive mRNA expression profiling to assess activation status, homing potential, and immunophenotype, is not possible on the immune cells recovered after infusion due to the small amount of recovered tissue. Fortunately, new technology is now available using the nCounter Analysis System from NanoString Technologies, Inc. (Seattle, WA). This system uses no enzymes or amplification steps, yet can analyze hundreds of genes in a single assay with high precision, even at low expression levels using samples directly obtained from cell culture, tissue, or whole blood lysates without sample purification and significantly can assay total purified RNA derived from formalin- fixed paraffin-embedded (FFPE) tissue samples. This system employs a novel digital technology to enable direct multiplexed measurement of gene expression while offering a high level of sensitivity and precision, including detection of fractional fold-change differences. The technology uses molecular barcodes and single molecule imaging to detect and count hundreds of unique transcripts in a single reaction. The fully automated system is easy to use and is ideally suited for researchers \on this grant application who wish to validate gene expression signatures, working with small amounts of starting material and studying defined gene sets.

描述（由申请人提供）： 基于细胞的免疫疗法对治疗恶性肿瘤有着巨大的希望，但它们是在现代医学中开发和实施的最大挑战之一。通常没有或很少的标记，例如不同的临床特征，成像的特定形态变化或血清学标识符，可以预测这些生物学疗法的功效甚至治疗潜力。尽管这些障碍以及从非营利性学术机构内部产生基于细胞的免疫疗法的挑战，但基于免疫的疗法仍在人类中开发和测试，基于人类的前提和承诺，其特异性和能力使用效应器机制使用不可超过传统治疗的效应器机制消除肿瘤的能力。德克萨斯大学（UT）M.D。Anderson癌症中心（MDACC）的癌症免疫学研究中心（CCIR）在设计和实施基于增强被动和主动免疫的临床级免疫疗法方面有一个积极的计划。当前，从患者（和动物）中恢复的细胞和细胞的表型的特征是流式细胞仪的蛋白质表达和对表达基因的有限分析。但是，至关重要的是，能够对免疫疗法前后差异表达的免疫基因进行分类，以评估治疗的治疗潜力，并进行下一代免疫治疗药的合理设计，并具有预测的生物学作用。当前的两种分析免疫基因表达的方法微分析和Q-PCR在增强由于所需的大量材料而引起的免疫疗法的潜力方面具有有限的作用，当mRNA被限制并且不涵盖阵列的动态浓度范围或伴有酶促扩增的偏见时，产生的产生偏差。简而言之，由于少量恢复的组织，在输注后回收的免疫细胞不可能进行全面的mRNA表达分析，以评估活化状态，归巢潜力和免疫表型。幸运的是，现在可以使用Nanostring Technologies，Inc。（华盛顿州西雅图）的NCounter分析系统获得新技术。该系统不使用酶或扩增步骤，但可以在单个测定中分析数百个基因，即使在低表达水平下，也可以使用直接从细胞培养，组织或全血裂解物获得的样品中分析，而没有样品纯化的整个血液裂解物，并且可以显着测定从福尔马林 - 固定固定的Paraffin-固定 - 固定 - 固定的 - 固定的 - 固定的 - 固定的 - 固定的 - 固定 - 固定 - 固定 - 固定的（ffppe）组织样品。该系统采用一种新型的数字技术来实现基因表达的直接多路复用测量，同时提供了高度的灵敏度和精确度，包括检测分数折叠变化差异。该技术使用分子条形码和单分子成像来检测并计算单个反应中数百个独特的转录本。完全自动化的系统易于使用，非常适合在此赠款应用程序上的研究人员\希望验证基因表达特征，使用少量的起始材料和研究定义的基因集。