Sensitive expression profiling in fixed archived tissue

固定存档组织中的敏感表达谱

• 批准号: 6997911
• 负责人: MARIANNA Mansfield GOLDRICK
• 金额: $ 26.4万
• 依托单位: AMBION, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6997911
• 关键词: DNA primers; RNA; RNase protection assay; biomarker; biotechnology; fluorescent dye /probe; gene expression profiling; human tissue; laboratory mouse; method development; microarray technology; neoplasm /cancer diagnosis; nucleic acid probes; nucleic acid quantitation /detection; polymerase chain reaction; tissue /cell preparation

DESCRIPTION (provided by applicant): Archives of formalin-fixed paraffin embedded (FFPE) human histological tissue samples, probably numbering in the millions of tissue blocks, constitute a tremendous, yet underutilized, historical resource for studying gene expression changes associated with human disease states. Unlike freshly acquired samples, there is usually greater documented medical history correlated to these archived specimens, including longterm treatment responses, adverse reactions, other complications. Therefore, they represent a potentially valuable source of RNA for use in expression profiling to identify markers for drug discovery and diagnostic and prognostic testing. However, they remain underutilized primarily because damage of RNA as a result of the fixation and embedding processes results in isolation of highly fragmented RNA from these samples. While quantitative real-time PCR (qRT-PCR) assays can tolerate fragmented RNA, sensitivity is substantially reduced. Sample-to-sample variations in the extent of fragmentation and potential biases in fragmentation between different mRNAs in a given sample further reduce the accuracy and reliability of qRT-PCR for FFPE samples. We have developed an assay that is particularly well-suited to the analysis of highly fragmented and damaged RNA to allow meaningful expression profiling from FFPE samples. Hybridization Amplification RNase Protection (HARP) uses chimeric DNA/RNA probes, containing RNA complementary to the target adjacent to DNA containing PCR primer sites. HARP probes are protected from RNase cleavage by hybridization with complementary target RNA and can be amplified using the DNA primer sites. The strength of the HARP assay is that the protected probe is longer than the short RNA target, and it is the probe, rather than the target, that is amplified and detected. This strategy is especially useful for detecting very short RNA targets, including those from FFPE samples. Our Phase I aims during are: 1) optimize the design of HARP probes for maximum signal to noise ratios and sensitivity in archived FFPE blocks, using qRT-PCR detection with dual-labeled fluorescent probes; 2) develop at least 20 HARP probes to individual targets associated with malignancy to be used for duplex real time PCR assays; and 3) adapt the probes generated in specific aim #2 so as to enable their simultaneous analysis using a liquid microbead array detection assay.

描述（由申请人提供）： 福尔马林固定石蜡嵌入（FFPE）人类组织学组织样本的档案，可能是数百万的组织块中的编号，构成了研究与人类疾病状态相关的基因表达变化的巨大但未充分利用的历史资源。与新鲜获得的样品不同，通常有更多的记录病史与这些存档标本有关，包括长期治疗反应，不良反应和其他并发症。因此，它们代表了用于表达分析的潜在有价值的RNA来源，以识别药物发现以及诊断和预后测试的标记。然而，它们仍然不足，主要是因为固定和嵌入过程导致RNA的损害导致从这些样品中分离出高度碎片的RNA。虽然定量的实时PCR（QRT-PCR）测定可以忍受碎片的RNA，但灵敏度大大降低。样本到样本的变化在给定样品中不同mRNA之间碎片化和潜在偏差的偏差进一步降低了QRT-PCR对FFPE样品的准确性和可靠性。我们开发了一种特别适合分析高度碎片和受损的RNA的测定法，以允许来自FFPE样品的有意义的表达分析。杂交扩增RNase Protection（HARP）使用嵌合DNA/RNA探针，其中包含与含有PCR启动位点的DNA相邻的靶标的RNA。通过与互补靶RNA杂交来保护HARP探针免受RNase裂解，并可以使用DNA引物位点扩增。竖琴测定法的强度是，受保护的探针比短RNA靶标长，并且被放大和检测到的探针而不是靶标。该策略对于检测非常短的RNA靶标特别有用，包​​括来自FFPE样品的靶标。我们的阶段I目标是：1）使用双标记的荧光探针使用QRT-PCR检测来优化存档FFPE块中最大信号与噪声比和灵敏度的设计； 2）至少将20个竖琴探针开发到与恶性肿瘤相关的个体靶标，用于双工实时PCR分析； 3）调整特定目标2中生成的探针，以便使用液体微粒阵列检测测定法实现其同时分析。