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。这项研究通过结合用于RNA Pol II转录本的新RNA纯化技术和下一代RNA测序来识别前哨RNA，并开发一种实用的生物样本RNA完整性检测方法，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。

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.