Sentinel Pol II RNAs for Measuring RNA Integrity in Biospecimens

用于测量生物样本中 RNA 完整性的 Sentinel Pol II RNA

• 批准号: 8078440
• 负责人: CURT H. HAGEDORN
• 金额: $ 21.86万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8078440
• 关键词: Affect; Biological Assay; Biological Markers; Breast; Budgets; Cancer Center; Cancer Patient; Clinical; Clinical Research; Clinical Trials; Code; Collection; Colon; Colon Carcinoma; DNA Polymerase III; Data; Deterioration; Diagnosis; Diagnostic; Diagnostic tests; Early Diagnosis; Electrophoresis; Ensure; Expenditure; FDA approved; Functional RNA; Future; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Genome; Goals; Human; International; Laboratory Procedures; Length; Liver; Malignant Neoplasms; Malignant neoplasm of liver; Mammary Gland Parenchyma; Measures; Medical; Medicare; Medicine; Messenger RNA; Molecular; Monitor; Outcome; Patient Care; Patients; Pattern; Polymerase; Preventive; Procedures; Process; Proteins; RNA; RNA Caps; RNA Degradation; RNA Polymerase II; RNA Sequences; RNA purification; RNA, Ribosomal, 18S; Relative (related person); Resolution; Reverse Transcription; Ribonucleases; Ribosomal RNA; Running; Sampling; Sentinel; Specimen; Technology; Temperature; Testing; Therapeutic; Time; Tissue Sample; Tissues; Transcript; United States; assay development; base; cohort; cost effective; data mining; genome-wide; improved; innovation; innovative technologies; mRNA Decay; mRNA Transcript Degradation; malignant breast neoplasm; new technology; next generation; novel strategies; outcome forecast; prognostic; response; sample collection; standard measure

DESCRIPTION (provided by applicant): The presence and quantity of specific RNA polymerase (Pol) II transcripts in biospecimens, that reflect gene expression patterns, are increasingly being used as biomarkers to make major patient care decisions (e.g., MammaPrintTM, array assay for 70 mRNAs). However, mRNA molecules in biospecimens are highly susceptible to degradation by RNases during sample collection, handling, and storage. Reliable data obtained by transcriptome analysis of biospecimens, with gene arrays or RNA-Sequencing, is highly dependent on the quality of the RNA analyzed. Current standard measures of RNA integrity focus on 28S and 18S ribosomal RNA. However, diagnostic and prognostic gene expression markers of cancer focus on changes in mRNAs which are RNA Pol II transcripts. The 5' ends of Pol II RNAs are unique in that they have a 5' m7GpppN cap. We will use new technologies to identify a panel of sentinel Pol II RNA transcripts in human colon, breast and liver biospecimens that mirror mRNA quality and use these RNAs to develop a 3'/5' PCR based assay that more accurately assesses the integrity of Pol II transcripts in biospecimens. We developed a new approach to identify and characterize all Pol II transcripts present in biospecimens by isolating 5' m7G capped RNAs and analyzing them with RNA-sequencing technologies (Illumina). This allows us to identify and quantitate both protein coding and non-coding regulatory RNAs in biospecimens. It also allows us to define the entire length of each RNA, define their 5'-3' pattern of degradation, and develop a qRT-PCR based assay of their 3' and 5' regions to measure their level of intactness. The degradation patterns of Pol II RNA transcripts will be assessed in freshly collected human colon, breast and liver biospecimens (normal and cancer) after increasing times at room temperature and commonly used handling procedures. This analysis will identify a panel of candidate sentinel RNAs that can be used to develop an assay for mRNA integrity in biospecimens. The Specific Aims are: 1) To identify candidate sentinel Pol II RNAs that mirror mRNA decay in specific biospecimens (see Example) and; 2) To develop a 5'/3' quantitative reverse transcription PCR (qRT-PCR) assay that measures the intactness of sentinel RNA transcripts in specific biospecimens to better measure mRNA integrity. This study stimulates technology innovation by combining a new RNA purification technology for RNA Pol II transcripts and next generation RNA-sequencing to identify sentinel RNAs and developing a practical assay for RNA integrity in biospecimens. Our studies will optimize the use of both currently stored and future collections of biospecimens in identifying gene expression biomarkers for the early diagnosis, prognosis, and response to therapy of cancer patients. The long-term goal of this technology is improving patient care and therapeutic outcomes by better determining the quality of RNA in biospecimens before conducting diagnostic or predictive gene expression tests. Two international experts in colon (Dr. Burt) and breast (Dr. Buys) cancer will provide expert advice during the development of this assay. PUBLIC HEALTH RELEVANCE: Thousands of clinical biospecimens are collected every year at cancer centers throughout the United States for the proper diagnosis and prognosis of breast, colon, and liver cancer. RNA molecules in these biospecimens are increasingly being used in diagnostic and prognostic testing and more recently in major therapeutic decisions. Unfortunately, the procedures for collection and storage of these biospecimens vary and many times not optimized to ensure sample integrity and quality. The purpose of this study is to develop an innovative technology to identify and assay a subset of mRNA molecules that are highly susceptible to degradation and can be used as molecular "sentinels" to better determine the quality of mRNA in clinical biospecimens before measuring panels of gene expression biomarkers. The long term goal of this study is improving patient care and therapeutic outcomes by better determining the quality of biospecimens before running multi gene expression diagnostic tests.

描述（由申请人提供）：生物样本中特定 RNA 聚合酶 (Pol) II 转录物的存在和数量反映了基因表达模式，越来越多地被用作生物标志物来做出主要的患者护理决策（例如，MammaPrintTM，针对 70 mRNA）。然而，生物样本中的 mRNA 分子在样品采集、处理和储存过程中极易被 RNase 降解。通过基因阵列或 RNA 测序对生物样本进行转录组分析获得的可靠数据高度依赖于所分析 RNA 的质量。目前 RNA 完整性的标准测量主要集中在 28S 和 18S 核糖体 RNA。然而，癌症的诊断和预后基因表达标记物集中于 mRNA 的变化，即 RNA Pol II 转录本。 Pol II RNA 的 5' 末端是独特的，因为它们具有 5' m7GpppN 帽。我们将使用新技术来鉴定人类结肠、乳房和肝脏生物样本中反映 mRNA 质量的一组前哨 Pol II RNA 转录本，并使用这些 RNA 开发基于 3'/5' PCR 的检测，更准确地评估 Pol II 的完整性。生物样本中的 II 转录本。我们开发了一种新方法，通过分离 5' m7G 加帽 RNA 并使用 RNA 测序技术 (Illumina) 对其进行分析，来识别和表征生物样本中存在的所有 Pol II 转录本。这使我们能够识别和定量生物样本中的蛋白质编码和非编码调节 RNA。它还使我们能够定义每个 RNA 的整个长度，定义其 5'-3' 降解模式，并开发基于 qRT-PCR 的 3' 和 5' 区域检测，以测量其完整性水平。在室温下增加时间和常用处理程序后，将在新鲜收集的人类结肠、乳房和肝脏生物样本（正常和癌症）中评估 Pol II RNA 转录本的降解模式。该分析将鉴定一组候选前哨 RNA，可用于开发生物样本中 mRNA 完整性的检测方法。具体目标是： 1) 鉴定反映特定生物样本中 mRNA 衰变的候选前哨 Pol II RNA（参见实施例）； 2) 开发一种 5'/3' 定量逆转录 PCR (qRT-PCR) 测定法，测量特定生物样本中前哨 RNA 转录物的完整性，以更好地测量 mRNA 完整性。这项研究通过结合用于 RNA Pol II 转录本的新型 RNA 纯化技术和下一代 RNA 测序来识别前哨 RNA，并开发了一种用于生物样本中 RNA 完整性的实用检测方法，从而刺激了技术创新。我们的研究将优化当前存储和未来收集的生物样本的使用，以识别基因表达生物标志物，以用于癌症患者的早期诊断、预后和治疗反应。该技术的长期目标是在进行诊断或预测基因表达测试之前更好地确定生物样本中 RNA 的质量，从而改善患者护理和治疗结果。结肠癌（Burt 博士）和乳腺癌（Buys 博士）的两位国际专家将在该检测的开发过程中提供专家建议。 公共健康相关性：美国各地的癌症中心每年都会收集数千份临床生物样本，用于乳腺癌、结肠癌和肝癌的正确诊断和预后。这些生物样本中的 RNA 分子越来越多地用于诊断和预后测试，最近还用于重大治疗决策。不幸的是，这些生物样本的收集和储存程序各不相同，而且很多时候都没有经过优化以确保样本的完整性和质量。本研究的目的是开发一种创新技术来识别和分析高度容易降解的 mRNA 分子子集，这些分子可用作分子“哨兵”，以便在测量基因组之前更好地确定临床生物样本中 mRNA 的质量表达生物标志物。这项研究的长期目标是通过在运行多基因表达诊断测试之前更好地确定生物样本的质量来改善患者护理和治疗结果。