Transcriptome profiling of highly degraded specimens through global analysis of short RNA fragments.

摘要 全球RNA测序技术彻底改变了健康和疾病基因调控的探索。 然而，RNA作为一种分子非常容易降解。因此，RNA测序， 在基础研究中成为常规，不能很好地与患者标本一起工作，因为它们的质量是可变的 并且不能总是被控制或预测。提高患者标本对转录组的适用性 分析，将开发一种用于RNA聚合酶II衍生的短RNA的全局分析的方法， 与现有的方法不同，它依赖于RNA降解，对低质量的RNA效果更好。 该方法基于酶促分离和短5 '-加帽RNA（scRNA）的全局测序。 片段，并被设计为与极低质量的RNA降解为短至20 - 30的片段一起工作。 五个核苷酸。这种低质量的样品目前不可能用任何现有方法进行分析。要求1 将建立导致最有效和特异性scRNA回收的步骤顺序，并将比较 新的程序对传统的RNA测序。目标2将确定样品降解的程度 当scRNA测序方法变得比常规RNA测序更有益时。测试 这项新技术将对以前从肺癌患者身上收集的保存标本进行分析 并将其基因表达谱与先前由 病理学家 这项工作将在整个RNA质量范围内建立癌症标本中RNA的无缝分析。 该程序预计将是足够简单的任何实验室在处理下一代 测序文库。这种新的能力将使实验室和测序核心设施能够接受高度 降解的样品，并进行降解和完整标本之间的全球转录组的比较。 该技术的更广泛应用将促进跨学科的合作，包括目前的 基础研究者和临床研究者之间缺乏联系 ! !

SUMMARY. Global RNA sequencing technologies have revolutionized exploration of gene regulation in health and disease. However, RNA as a molecule is highly susceptible to degradation. As a result, RNA-sequencing, which has become routine in basic research, does not work well with patient specimens because their quality is variable and can not be always controlled or predicted. To improve suitability of patient specimens for transcriptome profiling, an approach for global analysis of RNA polymerase II derived short RNAs will be developed that, unlike existing methods, relies on RNA degradation and works better for low-quality RNA. The approach is based on enzymatic isolation and global sequencing of short 5’-capped RNA (scRNA) fragments and is designed to work with extremely low quality RNA degraded to fragments as short as twenty- five nucleotides. Such low-quality samples are currently impossible to profile with any existing methods. Aim 1 will establish the order of steps that result in most efficient and specific scRNA recovery, and will compare the new procedure against conventional RNA-sequencing. Aim 2 will determine the extent of sample degradation when the scRNA sequencing approach becomes beneficial over conventional RNA sequencing. To test the new technology, profiling of preserved specimens previously collected from lung cancer patients will be done and their gene expression profiles will be tested against histological classification previously performed by a pathologist. The work will establish seamless profiling of RNA in cancer specimens across the entire RNA quality range. The procedure is expected to be sufficiently simple for any laboratory experienced in handling next-generation sequencing libraries. This new capability will enable laboratories and sequencing core facilities to accept highly degraded samples and perform comparison of global transcriptomes among degraded and intact specimens. Broader application of the technology will advance collaboration across disciplines including the presently tenuous connection between basic and clinical investigators. ! !