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

总结。 全球RNA测序技术已经彻底改变了对健康和疾病中基因调控的探索。 然而，RNA作为一种分子是非常容易降解的。因此，RNA测序，它具有 在基础研究中成为常规，不能很好地处理患者样本，因为它们的质量参差不齐 并且不能总是被控制或预测。提高患者标本转录组的适合性 为了对RNA聚合酶II衍生的短RNA进行全局分析，将开发一种方法， 与现有方法不同，该方法依赖于RNA降解，对低质量的RNA效果更好。 该方法基于酶法分离和短5‘-帽RNA(ScRNA)的全球测序。 片段，设计用于处理质量极低的RNA，这些片段被降解为短至20个 五个核苷酸。这种低质量的样品目前不可能用任何现有的方法进行分析。目标1 将建立导致最有效和最具体的scRNA回收的步骤顺序，并将比较 与传统的RNA测序相反的新程序。目标2将确定样品退化的程度 当单链RNA测序方法变得比传统的RNA测序方法更有利时。要测试 新技术，将对以前从肺癌患者那里收集的保存样本进行剖析 他们的基因表达谱将与之前由 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的无缝图谱。 对于任何在处理下一代技术方面有经验的实验室来说，这一过程都将非常简单 测序文库。这一新能力将使实验室和测序核心设施能够高度接受 并在退化和完整样本之间进行全局转录本的比较。 该技术的更广泛应用将促进跨学科的合作，包括目前 基础和临床研究人员之间的联系微乎其微。 好了！ 好了！

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. ! !