Standardized NanoArray PCR for Gene Expression Profiling of Lung Cancer

用于肺癌基因表达谱分析的标准化纳米阵列 PCR

• 批准号: 8005977
• 负责人: James C. Willey
• 金额: $ 15.38万
• 依托单位: UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-07 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8005977
• 关键词: Address; Adoption; Biological Assay; Cancer Prognosis; Characteristics; Chemistry; Clinical; Color; Consumption; Detection; Development; Diagnostic; Diagnostic Procedure; Diagnostic tests; Dyes; End Point Assay; Excision; Formalin; Freezing; Funding; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic Transcription; Genomics; Goals; Health; Label; Laboratories; Lead; Liquid substance; Lung; Lung Neoplasms; Malignant Neoplasms; Malignant neoplasm of lung; Measurement; Measures; Methods; Molecular; Molecular Diagnostic Testing; Molecular Profiling; Monitor; Morphologic artifacts; Nanoarray Analytical Device; Operative Surgical Procedures; Outcome; Paraffin Embedding; Performance; Phase; Quality Control; RNA; Recording of previous events; Reproducibility; Running; Sampling; Selection for Treatments; Site; Solutions; Specimen; Speed; Staging; System; Taq Polymerase; Technology; Testing; Therapeutic; Time; Universities; Validation; base; cost; design; flexibility; improved; inhibitor/antagonist; innovation; malignant breast neoplasm; meetings; nano; nanofluidic; neoplastic; novel; nuclease; outcome forecast; prognostic; research study; tumor; validation studies

DESCRIPTION (provided by applicant): Molecular characterization of cancer, in particular by gene transcription profiling, has great potential to improve prognosis, therapeutic selection and clinical outcomes. However, the potential for using expression signatures for cancer prognosis and treatment selection is hampered by lack of readily deployable test kits with the accuracy, low RNA requirement and inter-site concordance required for routine clinical use. We propose an innovative solution based on two well-validated PCR technologies whose combination uniquely addresses the problem of diagnostic assay reproducibility. Our plan is to implement Standardized RT (StaRT)-PCR, a proven competitive PCR method developed at the University of Toledo, in a novel nanofluidic PCR platform developed by BioTrove Inc. in order to streamline the fluidic workflow, improve measurement throughput, and at the same time reduce test cost and maintain low RNA input. As compared to existing hybridization or real-time qPCR approaches, Standardized NanoArray PCR (SNAP) will provide the same dynamic range and quality as RT-PCR, yet require less RNA input and be more readily clinically deployable. The development will entail step-wise integration of proven technologies. First, real-time qPCR TaqMan assays will be developed for 16 lung tumor prognostic genes. These assays will be converted to StaRT-PCR by creation of competitive template and a competitor specific dye-labeled probe. Adding a pre-amplification step to StaRT-PCR will reduce the RNA input requirements to enable thousands of tests per sample. Finally, moving the assays into the OpenArray nano-PCR plate will streamline fluid handling. Using RNA isolated from lung clinical tumor resections, dynamic range and precision equivalent to real-time qPCR will be demonstrated for the integrated platform. After the initial development phase is complete, we will compare SNAP and real time qPCR in two critical gene expression profiling experiments. First we will compare the minimum amount of RNA required for each method by monitoring loss of precision as a function of decreasing RNA sample input. Second we will demonstrate lower inter-site variability, a critical factor for deploy-ability, by measuring gene expression profiles of seven lung tumor resection samples in three laboratories. Meeting these Specific Aims will lead to seeking further funding for multi-site prognostic validation studies involving formalin-fixed, paraffin-embedded (FFPE) lung specimens with extensive clinical history.

描述（由申请人提供）：癌症的分子表征，特别是通过基因转录谱分析，具有改善预后、治疗选择和临床结果的巨大潜力。然而，由于缺乏具有常规临床使用所需的准确性、低RNA要求和位点间一致性的易于部署的测试试剂盒，使用表达标记进行癌症预后和治疗选择的潜力受到阻碍。我们提出了一个创新的解决方案，基于两个经过验证的PCR技术，其组合独特地解决了诊断检测重现性的问题。我们的计划是在BioTrove Inc.开发的新型纳米流体PCR平台中实施标准化RT（StaRT）-PCR，这是托莱多大学开发的一种经过验证的竞争性PCR方法。以简化流体工作流程，提高测量通量，同时降低测试成本并保持低RNA输入。与现有的杂交或实时qPCR方法相比，标准化NanoArray PCR（SNAP）将提供与RT-PCR相同的动态范围和质量，但需要更少的RNA输入，并且更容易在临床上部署。开发将需要逐步整合经过验证的技术。首先，将针对16个肺肿瘤预后基因开发实时qPCR TaqMan测定。通过创建竞争性模板和竞争对手特异性染料标记探针，将这些检测转化为StaRT-PCR。在StaRT-PCR中增加预扩增步骤将减少RNA输入要求，从而使每个样本能够进行数千次测试。最后，将测定移至OpenArray nano-PCR板中将简化流体处理。使用从肺临床肿瘤切除中分离的RNA，将证明集成平台的动态范围和精度等同于实时qPCR。初始开发阶段完成后，我们将在两项关键基因表达谱分析实验中比较SNAP和真实的时间qPCR。首先，我们将通过监测作为RNA样品输入减少的函数的精密度损失来比较每种方法所需的RNA的最小量。其次，我们将通过测量三个实验室中七个肺肿瘤切除样本的基因表达谱来证明较低的部位间变异性，这是部署能力的关键因素。满足这些特定目标将导致寻求更多资金用于多中心预后验证研究，涉及具有广泛临床病史的福尔马林固定石蜡包埋（FFPE）肺标本。

项目成果

Control for stochastic sampling variation and qualitative sequencing error in next generation sequencing.

• DOI: 10.1016/j.bdq.2015.08.003
• 发表时间: 2015-09-01
• 期刊: Biomolecular detection and quantification
• 作者: Blomquist T;Crawford EL;Yeo J;Zhang X;Willey JC
• 通讯作者: Willey JC

A multiplex two-color real-time PCR method for quality-controlled molecular diagnostic testing of FFPE samples.

• DOI: 10.1371/journal.pone.0089395
• 发表时间: 2014
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Yeo J;Crawford EL;Blomquist TM;Stanoszek LM;Dannemiller RE;Zyrek J;De Las Casas LE;Khuder SA;Willey JC
• 通讯作者: Willey JC