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

基本信息

批准号：
8325038
负责人：
CURT H. HAGEDORN
金额：
$ 12.47万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8325038
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

项目摘要

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.

描述（由申请人提供）：反映基因表达模式的特定RNA聚合酶（POL）II转录本的存在和数量越来越多地被用作生物标志物，以做出重大的患者护理决策（例如Mammaprinttm，阵列，阵列分析70 mRNA）。然而，生物测量中的mRNA分子在采集，处理和存储期间高度易于RNase降解。通过基因阵列或RNA序列的生物测量的转录组分析获得的可靠数据高度取决于所分析的RNA的质量。 RNA完整性的当前标准度量将重点放在28S和18S核糖体RNA上。然而，癌症的诊断和预后基因表达标志物集中于RNA Pol II转录本的mRNA变化。 Pol II RNA的5'端是独一无二的，因为它们具有5'M7GPPPN盖。我们将使用新技术来识别人类结肠，乳腺癌和肝脏生物测量的一组Sentinel Pol II RNA转录本，这些转录镜像mRNA质量，并使用这些RNA来开发基于3'/5'的PCR测定法，从而更准确地评估POL II在生物生物测量中POL II转录的完整性。我们开发了一种新的方法来识别和表征生物测量中存在的所有POL II转录本，通过隔离5'M7G限制的RNA并使用RNA测序技术（Illumina）分析它们。这使我们能够识别和量化生物测量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，定义其5'-3'降解模式，并开发基于QRT-PCR的3'和5'区域的基于QRT-PCR的测定，以测量其完整性水平。 POL II RNA转录本的降解模式将在室温和常用处理程序的新鲜人类结肠，乳腺癌和肝脏生物测量（正常和癌症）中进行评估。该分析将确定一组候选Sentinel RNA，该小组可用于开发生物测量中mRNA完整性的测定法。具体目的是：1）确定在特定的生物测量中反映mRNA衰减的候选哨兵pol II RNA（见示例）和； 2）开发一个5'/3'定量逆转录PCR（QRT-PCR）测定，该测定法测量了特定生物测量中前哨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成绩单的新RNA纯化技术和下一代RNA测序来刺激技术创新，以鉴定Sentinel RNA并开发生物测量中RNA完整性的实用测定法。我们的研究将优化目前存储的生物测量和未来收集生物种子的收集，以鉴定基因表达生物标志物，以早期诊断，预后和对癌症患者治疗的反应。该技术的长期目标是通过在进行诊断或预测基因表达测试之前更好地确定生物测量中的RNA质量来改善患者护理和治疗结果。在开发此测定期间，两名国际科隆（Burt）和乳腺癌（Buys）癌症（Buys）癌症的国际专家将提供专家建议。