Sensitive expression profiling in fixed archived tissue

固定存档组织中的敏感表达谱

• 批准号: 7214379
• 负责人: GARY J LATHAM
• 金额: $ 24.69万
• 依托单位: AMBION DIAGNOSTICS, 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 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7214379
• 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）： 福尔马林固定石蜡包埋 (FFPE) 人类组织学组织样本档案（数量可能有数百万个组织块）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得到充分利用的历史资源。与新采集的样本不同，这些存档样本通常有更多记录的病史，包括长期治疗反应、不良反应和其他并发症。因此，它们代表了一种潜在有价值的 RNA 来源，可用于表达谱分析，以识别药物发现、诊断和预后测试的标记。然而，它们仍未得到充分利用，主要是因为固定和嵌入过程导致 RNA 损伤，导致从这些样品中分离出高度碎片化的 RNA。虽然定量实时 PCR (qRT-PCR) 检测可以耐受片段化 RNA，但灵敏度会大幅降低。样品之间的片段化程度差异以及给定样品中不同 mRNA 之间片段化的潜在偏差进一步降低了 FFPE 样品的 qRT-PCR 的准确性和可靠性。我们开发了一种特别适合分析高度片段化和受损的 RNA 的检测方法，以便对 FFPE 样品进行有意义的表达谱分析。杂交扩增 RNase 保护 (HARP) 使用嵌合 DNA/RNA 探针，其中包含与含有 PCR 引物位点的 DNA 相邻的靶标互补的 RNA。 HARP 探针通过与互补靶 RNA 杂交而免受 RNase 切割，并且可以使用 DNA 引物位点进行扩增。 HARP 检测的优势在于受保护的探针比短 RNA 靶标更长，并且被扩增和检测的是探针，而不是靶标。该策略对于检测非常短的 RNA 靶标（包括来自 FFPE 样品的 RNA 靶标）特别有用。我们第一阶段的目标是：1) 使用双标记荧光探针进行 qRT-PCR 检测，优化 HARP 探针的设计，以实现存档 FFPE 块中的最大信噪比和灵敏度； 2) 针对与恶性肿瘤相关的单个靶标开发至少 20 个 HARP 探针，用于双重实时 PCR 检测； 3) 调整在特定目标#2中生成的探针，以便能够使用液体微珠阵列检测测定对其进行同步分析。