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）癌症组织中基因表达的定量对于研究癌症的许多方面非常重要，包括肿瘤的发生、其分类、进展和对治疗的反应性，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灵敏度和序列特异性，RT-PCR方法经常用于mRNA表达谱分析和验证微阵列获得的数据。然而，在FFPE样品的制备和储存期间发生的RNA随机片段化降低了RT-qPCR的灵敏度和再现性。为了克服这个问题，我们提出了一种新的方法，用于测定mRNA片段在FFPE样品称为mR-FQ（mRNA片段定量），提供了上级的灵敏度和准确性，尽管在一个具有成本效益的方式片段。我们的新方法引入了三项关键创新。首先，我们纯化一定大小范围的RNA片段。第二，我们以允许更有效的逆转录和靶mRNA序列的同时预扩增的方式修饰（片段化的）mRNA。第三，我们使用了一种新的PCR引物设计，该设计提供了上级灵敏度和特异性，同时使用单一染料读出（例如，SYBR绿色），而不是专门的探针如TaqMan探针。在第一阶段，我们将为这种新的检测方法建立概念验证，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RTR-qPCR检测方法相比具有上级灵敏度。在第二阶段，我们将为所有已建立的乳腺癌mRNA生物标志物开发额外的检测方法。然后，我们将开发一种虚拟PCR阵列形式的检测方法，用于FFPE样本中mRNA和miRNA生物标志物的同时表达谱分析。（测定两种类型的RNA将提供比仅测定一种类型的RNA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，基于该检测方法共同开发乳腺癌伴随诊断检测方法。将这种检测方法应用于临床可能对患者有益，并可能改善临床决策。 公共卫生相关性：乳腺癌是女性中最常见的癌症。仅在美国，去年就有大约20万名妇女被诊断出患有这种疾病，超过4万人死于这种疾病。本基金申请的目标是开发一种方法，用于定量标准组织学标本（FFPE块）中的片段mRNA，该方法将以合理的成本提供上级灵敏度和准确性，以促进乳腺癌的研究和诊断。

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.