Sentinel Pol II RNAs for Measuring RNA Integrity in Biospecimens

描述（由申请人提供）：生物标本中特异性RNA聚合酶（Pol） II转录本的存在和数量反映了基因表达模式，越来越多地被用作主要患者护理决策的生物标志物（例如，MammaPrintTM， 70个mrna的阵列分析）。然而，生物标本中的mRNA分子在样品收集、处理和储存过程中极易被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完整性的标准测量主要集中在28S和18S核糖体RNA上。然而，癌症的诊断和预后基因表达标志物主要集中在RNA Pol II转录物mrna的变化上。Pol II RNA的5‘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鉴定一组反映mRNA质量的前哨Pol II RNA转录本，并使用这些RNA开发基于3'/5' PCR的检测方法，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来鉴定和表征生物标本中存在的所有Pol II转录本，方法是分离5' m7G封顶rna，并使用rna测序技术（Illumina）对其进行分析。这使我们能够在生物标本中识别和定量蛋白质编码和非编码调控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’区域检测来测量它们的完整性水平。在室温和常用处理程序下增加时间后，将在新鲜收集的人类结肠、乳腺和肝脏生物标本（正常和癌症）中评估Pol II RNA转录物的降解模式。该分析将确定一组候选前哨rna，可用于开发生物标本中mRNA完整性的测定。具体目的是：1)鉴定反映特定生物标本中mRNA衰变的候选前哨Pol II rna（见示例）；2)建立5‘/3’定量反转录PCR （qRT-PCR）检测方法，检测特定生物标本中前哨RNA转录物的完整性，以更好地测量mRNA的完整性。本研究通过结合RNA Pol II转录本的新RNA纯化技术和下一代RNA测序来鉴定前哨RNA，并开发生物标本中RNA完整性的实用测定方法，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标本的使用，以确定基因表达生物标志物，用于癌症患者的早期诊断、预后和对治疗的反应。这项技术的长期目标是在进行诊断或预测基因表达测试之前，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效果。两位国际结肠癌专家（Dr. Burt）和乳腺癌专家（Dr. Buys）将在这项检测的开发过程中提供专家建议。

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.