Sentinel Pol II RNAs for Measuring RNA Integrity in Biospecimens

描述(由申请人提供)：生物标本中特定的RNA聚合酶(POL)II转录本的存在和数量反映了基因表达模式，越来越多地被用作做出重大患者护理决策的生物标记物(例如，MammaPrintTM，70mRNAs的阵列分析)。然而，在样本采集、处理和储存过程中，生物旋毛虫中的信使核糖核酸分子极易被核酸酶降解。通过基因阵列或RNA测序对生物标本进行转录组分析所获得的可靠数据，高度依赖于所分析的RNA的质量。目前衡量RNA完整性的标准标准集中在28S和18S核糖体RNA上。然而，癌症的诊断和预后基因表达标记关注的是作为RNA POLII转录本的mRNAs的变化。Pol II RNA的5‘末端是独一无二的，因为它们有一个5’m7GpppN帽。我们将使用新技术在人类结肠、乳房和肝脏生物标本中识别一组反映mRNA质量的前哨POL II RNA转录本，并使用这些RNA开发一种基于3‘/5’PCR的分析方法，更准确地评估生物标本中POL II转录本的完整性。我们开发了一种新的方法，通过分离5‘m7G封端的RNA并使用RNA测序技术(Illumina)对其进行分析，从而鉴定和鉴定存在于生物检疫菌中的所有POL II转录本。这使我们能够识别和定量生物检疫菌中的蛋白质编码和非编码调控RNA。它还允许我们定义每个RNA的全长，定义它们的5‘-3’降解模式，并建立基于qRT-PCR的3‘和5’区域的分析方法，以测量它们的完整性水平。在室温下增加时间和常用的处理程序后，将在新收集的人类结肠、乳腺和肝脏生物标本(正常和癌症)中评估Pol II RNA转录本的降解模式。这项分析将确定一组候选的前哨RNA，这些RNA可用于开发一种检测生物标本中mRNA完整性的方法。具体目的是：1)确定反映特定生物样本中mRNA衰退的候选前哨Pol II RNA；2)建立一种5‘/3’定量逆转录聚合酶链式反应(qRT-PCR)方法，测量特定生物样本中前哨RNA转录本的完整性，以更好地衡量mRNA的完整性。这项研究通过将一种新的RNA POL II转录本的RNA纯化技术和下一代RNA测序相结合来识别前哨RNA，并开发了一种实用的生物检疫菌RNA完整性检测方法，从而刺激了技术创新。我们的研究将优化目前储存和未来收集的生物样本的使用，以确定癌症患者早期诊断、预后和治疗反应的基因表达生物标记物。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(Dr.Buys)方面的国际专家将在该检测方法的开发过程中提供专家建议。 公共卫生相关性：美国各地的癌症中心每年收集数以千计的临床生物标本，用于乳腺癌、结肠癌和肝癌的正确诊断和预后。这些生物样品中的RNA分子越来越多地被用于诊断和预后测试，最近还被用于重大治疗决策。不幸的是，这些生物标本的收集和储存程序各不相同，而且许多时候没有优化以确保样本的完整性和质量。这项研究的目的是开发一种创新的技术来识别和分析一组高度易降解的、可用作分子“哨兵”的mRNA分子子集，以便在测量基因表达生物标记物面板之前更好地确定临床生物标本中的mRNA质量。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标本的质量来改善患者护理和治疗结果。

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.