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

一种定量 mRNA 片段的新方法 (mR-FQ)

• 批准号: 8250879
• 负责人: SERGEI A KAZAKOV
• 金额: $ 30万
• 依托单位: SOMAGENICS, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-21 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8250879
• 关键词: 7SL RNA; Applications Grants; Biochemical Reaction; Biological; Biological Assay; Biological Markers; Classification; Clinic; Collaborations; Companions; Complementary DNA; DNA; DNA-Directed DNA Polymerase; Data; Dependence; Detection; Development; Diagnosis; Diagnostic; Disease; Dyes; ERBB2 gene; Evaluation; Exonuclease; Formalin; Gel; Gene Expression; Genes; Goals; Histology; In Vitro; Length; Ligase; Malignant Neoplasms; Messenger RNA; Methods; MicroRNAs; Molecular Profiling; Paraffin Embedding; Patients; Performance; Phase; Preparation; Procedures; RNA; RNA Sequences; RNA-Directed DNA Polymerase; Reaction; Relative (related person); Reproducibility; Research; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Sampling; Sensitivity and Specificity; Specificity; Specimen; Testing; Tissue Sample; Tissues; Transcript; Validation; Variant; Woman; Work; anticancer research; base; breast cancer diagnosis; cancer therapy; circular RNA; clinical decision-making; cost; cost effective; design; gel electrophoresis; improved; innovation; internal control; mRNA Expression; malignant breast neoplasm; novel; novel strategies; prognostic; tumor; tumor initiation; two-dimensional; virtual

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.

描述（由申请人提供）：在福尔马林固定石蜡包埋（FFPE）癌症组织中对基因表达的定量对于研究癌症的许多方面，包括肿瘤的启动，其分类，对治疗的反应和治疗的反应以及将特定的特定基因作为诊断和/或预后生物标志物验证。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表达分析和通过微阵列获得的验证数据。但是，在FFPE样品制备和存储过程中发生的RNA的随机片段降解了RT-QPCR的灵敏度和可重复性。为了克服这个问题，我们提出了一种新的方法，用于在称为MR-FQ（mRNA片段定量）的FFPE样品中分析mRNA片段，尽管以经济有效的方式进行了片段化，该方法具有较高的灵敏度和准确性。我们的新方法介绍了三个关键的创新。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靶mRNA序列的方式来修改（碎片）mRNA。第三，我们使用一种新型的PCR引物设计，该设计在使用单DYE读数（例如，使用SYBR绿色）的同时提供了卓越的灵敏度和特异性，而不是Taqman探针等专业探针。在第一阶段，我们将建立该新测定法的概念概念，将其应用于乳腺癌FFPE组织样品中HER-2 mRNA的检测，并证明其与常规RTR-QPCR分析相比，它具有出色的敏感性。在第二阶段，我们将为所有已建立的mRNA生物标志物用于乳腺癌的其他测定。然后，我们将以虚拟PCR阵列格式开发一种测定法，以在FFPE样品中同时对mRNA和miRNA生物标志物的同时表达分析。 （分析两类RNA将比仅分析一种类型的RNA提供更可靠的生物标志物）。我们还将通过与领先的制药公司和诊断公司的合作，基于该测定法共同开发乳腺癌伴侣诊断测定法。将此测定法进入诊所可能对患者有益，并有可能改善临床决策。 公共卫生相关性：乳腺癌是女性中最常见的癌症。去年，仅在美国，大约有20万名妇女被诊断出，超过40,000名死于该疾病。该赠款应用的目的是开发一种定量标准组织学标本（FFPE块）碎片mRNA的方法，该方法将以合理的成本提供较高的敏感性和准确性，以促进乳腺癌研究和诊断。