Sensitive expression profiling in fixed archived tissue

点击翻译按钮获取中文摘要

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.