Sensitive expression profiling in fixed archived tissue

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的档案，可能有数百万个组织块，构成了一个巨大的，但未充分利用，研究与人类疾病状态相关的基因表达变化的历史资源。与新鲜采集的样本不同，通常有更多与这些存档标本相关的记录病史，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的有价值的RNA来源，可用于表达谱分析，以鉴定用于药物发现和诊断及预后测试的标记物。然而，它们仍然未被充分利用，主要是因为固定和包埋过程导致的RNA损伤导致从这些样品中分离出高度片段化的RNA。虽然定量实时PCR（qRT-PCR）测定可以耐受片段化的RNA，但灵敏度显著降低。片段化程度的样品间差异和给定样品中不同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RNA的测定法，以允许从FFPE样品进行有意义的表达谱分析。杂交扩增RNA酶保护（HARP）使用嵌合DNA/RNA探针，其含有与含有PCR引物位点的DNA相邻的靶互补的RNA。HARP探针通过与互补靶RNA杂交而被保护免受RNA酶切割，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靶标长，并且是探针而不是靶标被扩增和检测。这种策略对于检测非常短的RNA靶标特别有用，包括来自FFPE样品的那些。我们的I期目标是：1）使用双标记荧光探针的qRT-PCR检测，优化HARP探针的设计，以在存档的FFPE块中获得最大的信噪比和灵敏度; 2）开发至少20种针对与恶性肿瘤相关的个体靶标的HARP探针，以用于双重真实的时间PCR测定;和3）调整在特定目标#2中产生的探针，以便能够使用液体微珠阵列检测测定法对其进行同时分析。

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.