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）：生物标本中特异性RNA聚合酶（Pol）II转录物的存在和数量反映了基因表达模式，正越来越多地用作生物标志物，以做出重大的患者护理决策（例如，MammaPrintTM，70种mRNA的阵列测定）。然而，生物样本中的mRNA分子在样本采集、处理和储存期间极易被RNA酶降解。通过基因阵列或RNA测序对生物标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完整性的标准测量集中在28 S和18 S核糖体RNA上。然而，癌症的诊断和预后基因表达标志物集中在mRNA的变化，即RNA Pol II转录物。Pol II RNA的5'端是独特的，因为它们具有5' m7 GpppN帽。我们将使用新技术来鉴定人类结肠、乳腺和肝脏生物标本中反映mRNA质量的一组前哨Pol II RNA转录物，并使用这些RNA开发基于3 '/5' PCR的检测方法，以更准确地评估生物标本中Pol II转录物的完整性。我们开发了一种新的方法来鉴定和表征存在于生物标本中的所有Pol II转录物，通过分离5'm7 G加帽RNA并用RNA测序技术（Illumina）分析它们。这使我们能够识别和定量生物标本中的蛋白质编码和非编码调节RNA。它还允许我们定义每个RNA的整个长度，定义它们的5 '-3'降解模式，并开发基于qRT-PCR的3'和5'区域测定以测量它们的完整性水平。在室温下增加时间和常用处理程序后，将在新鲜采集的人结肠、乳腺和肝脏生物标本（正常和癌症）中评估Pol II RNA转录物的降解模式。该分析将确定一组候选哨兵RNA，可用于开发生物标本中mRNA完整性的测定。具体目标是：1）鉴定反映特定生物样本中mRNA衰变的候选哨兵Pol II RNA（参见实施例）; 2）开发测量特定生物样本中哨兵RNA转录物的完整性以更好地测量mRNA完整性的5 '/3'定量逆转录PCR（qRT-PCR）测定。本研究通过结合新的RNA纯化技术用于RNA Pol II转录本和下一代RNA测序来识别哨兵RNA，并开发生物标本中RNA完整性的实用检测方法，从而刺激技术创新。我们的研究将优化使用目前储存和未来收集的生物标本，以确定基因表达生物标志物，用于癌症患者的早期诊断，预后和治疗反应。这项技术的长期目标是在进行诊断或预测性基因表达测试之前，通过更好地确定生物标本中RNA的质量来改善患者护理和治疗结果。两位结肠癌（Burt博士）和乳腺癌（Buys博士）国际专家将在本检测试剂盒开发期间提供专家建议。 公共卫生相关性：每年在美国各地的癌症中心收集数千份临床生物标本，用于乳腺癌、结肠癌和肝癌的正确诊断和预后。这些生物标本中的RNA分子越来越多地用于诊断和预后检测，最近还用于重大治疗决策。不幸的是，这些生物样本的采集和储存程序各不相同，而且很多时候没有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，以识别和测定一个非常容易降解的mRNA分子子集，并可用作分子“哨兵”，以更好地确定临床生物标本中mRNA的质量，然后再测量基因表达生物标志物。本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。