A novel method for quantitation of fragmented mRNAs (mR-FQ)

描述（由申请人提供）：福尔马林固定石蜡包埋（FFPE）癌组织中基因表达的定量对于研究癌症的许多方面都很重要，包括肿瘤的发生、分类、进展和对治疗的反应，以及验证特定基因作为诊断和/或预后生物标志物。由于其令人印象深刻的敏感性和序列特异性，RT-PCR方法经常用于mRNA表达谱和验证微阵列获得的数据。然而，在制备和储存FFPE样品过程中发生的随机RNA断裂降低了RT-qPCR的灵敏度和再现性。为了克服这一问题，我们提出了一种分析FFPE样品中mRNA片段的新方法，称为mR-FQ （mRNA片段量化），该方法以经济有效的方式提供了优越的灵敏度和准确性，尽管碎片化。我们的新方法引入了三个关键创新。首先，我们纯化一定大小范围的RNA片段。其次，我们以一种允许更有效的逆转录和同时预扩增目标mRNA序列的方式修改（碎片化）mRNA。第三，我们使用了一种新颖的PCR引物设计，在使用单染料读数（例如，SYBR Green）时提供了优越的灵敏度和特异性，而不是专门的探针，如TaqMan探针。在I期，我们将建立这种新方法的概念验证，将其应用于乳腺癌FFPE组织样本中HER-2 mRNA的检测，并证明其与传统的RTR-qPCR方法相比具有更高的灵敏度。在II期，我们将开发所有已建立的乳腺癌mRNA生物标志物的附加检测。然后，我们将开发一种虚拟pcr阵列格式的检测方法，用于同时分析FFPE样品中mRNA和miRNA生物标志物的表达。（分析这两类RNA比只分析一种RNA提供更可靠的生物标志物）。我们还将通过与领先的制药和诊断公司合作，共同开发基于该检测的乳腺癌伴随诊断检测。将这种检测方法应用于临床可能对患者有益，并有可能改善临床决策。

DESCRIPTION (provided by applicant): Quantitation of gene expression in formalin-fixed paraffin-embedded (FFPE) cancer tissues is important for studying many aspects of cancer, including the initiation of tumors, their classification, progression and responsiveness to treatment, and for validating specific genes as diagnostic and/or prognostic biomarkers. Due to their impressive sensitivity and sequence specificity, RT-PCR methods are frequently used for mRNA expression profiling and for validating data obtained by microarrays. However, the random fragmentation of RNA that occurs during preparation and storage of FFPE samples degrades the sensitivity and reproducibility of RT-qPCR. To overcome this problem, we propose a novel method for assaying mRNA fragments in FFPE samples called mR-FQ (mRNA Fragment Quantification) that provides superior sensitivity and accuracy in spite of fragmentation in a cost-effective manner. Our new approach introduces three key innovations. First, we purify RNA fragments of a certain size range. Second, we modify the (fragmented) mRNA in a way that allows for more efficient reverse transcription and simultaneous pre-amplification of target mRNA sequences. Third, we use a novel PCR primer design that provides superior sensitivity and specificity while using single-dye readout (e.g., with SYBR Green), as opposed to specialized probes such as TaqMan probes. In Phase I, we will establish proof-of-concept for this new assay, apply it for detection of HER-2 mRNA in breast cancer FFPE tissue samples, and demonstrate its superior sensitivity in comparison to conventional RTR-qPCR assays. In Phase II, we will develop additional assays for all established mRNA biomarkers for breast cancer. Then, we will develop an assay in virtual PCR-array format for simultaneous expression profiling of both mRNA and miRNA biomarkers in FFPE samples. (Assaying both classes of RNA would provide a more reliable biomarker than assaying only one type of RNA). We will also co-develop breast cancer companion diagnostic assays based on this assay through collaboration with leading Pharma and diagnostic companies. Taking this assay into the clinic could be beneficial for patients and potentially improve clinical decision-making. PUBLIC HEALTH RELEVANCE: Breast cancer is the most common cancer among women. About 200,000 women were diagnosed and over 40,000 died from the disease last year in the US alone. The goal of this grant application is to develop a method for quantitation of fragmented mRNAs from standard histology specimens (FFPE blocks) that will provide superior sensitivity and accuracy at a reasonable cost to facilitate research in and diagnosis of breast cancer.