The Pain Neural Transcriptome

摘要：该项目正处于起步阶段。至此，我们已经建立了几种研究方法和协议，在设备、实验室空间、协作安排等方面建立了配套基础设施，聘请并开始培训一支科学家团队和支持人员来进行研究项目。在今年的过程中，我们在Illumina HiSeq 2000上进行了150多次测序，并检查了外周炎症、周围神经损伤、关节炎模型、基因标记的神经元群体的测序，并完成了几个物种的三叉神经转录组。我们正在就这些数据编写多份报告。一些最有趣的观察与确切的基因表达有关，这些基因介导了已知止痛药如吗啡、可乐定以及钠和钙通道阻滞剂的作用。我们现在可以指定哪个伤害性神经元群体是由哪种止痛药控制的，因为我们正在研究模式物种和人类死后组织，我们可以推广到人类，并将止痛研究集中在最相关的已知和新的分子靶点上。 这些人员现在包括一名受过生物化学和生物信息学培训的博士研究员，一名受过生物信息学和计算生物学培训的博士后研究员，以及一名在RNA提取方面有经验的BAC后IRTA，他还将接受cDNA文库构建和RT-PCR分析方面的培训。该实验室购买了两台用于高速序列比对和转录组文库分析的专用计算机工作站，并建造了第三台。我们升级了我们的生物分析仪，用于RNA质量评估，并利用96和384个良好的实时聚合酶链式反应设备来独立验证RNA-SEQ方法中看到的mRNA变化。对于测序，我们与美国国家研究院测序中心(NISC)的研究人员合作，主要使用Illumina High-Seq 2000平台进行深度测序，通常以多重模式分析6个独立样本/车道。我们确定，在构建测序文库时，Polya+选择优于核糖体RNA耗竭。我们与宾夕法尼亚大学签订了合同，为RNA研究获取犬类组织。这些组织将从对照组和因疼痛控制不充分而被安乐死或接受树脂毒素治疗的骨肉瘤动物身上获得，以及在尸检中获得的组织。获得的数据将使我们能够测试由自然发生的骨癌的伤害性输入激活的基因，并通过治疗进行调节。在犬类研究中的收益接近完成。这些数据还将用于与在小鼠、大鼠和人类身上进行的平行研究进行比较(尽管确切的模型将有所不同)。 转录组项目正在系统地研究疼痛通路的前三个步骤，从受损的外周组织或神经、背根神经节和背(感觉)脊髓开始。面部和头部的等效结构是三叉神经节和延髓(延髓背角)。我们与NIMH的Joel Kleinman博士合作获得并分析了这两种组织。这些相同的组织将从老鼠、狗和猴子身上进行分析。利用生物分析仪对人延髓背角和三叉神经节的总RNA进行了分析。经过几次技术改进后，分析得到了极好的RNA完整性数字。我们几乎完成了对大鼠炎症诱导基因的初步研究。这包括对发炎的外周组织、背根神经节和背角中基因调控的时间进程的系统检查。将对几种神经损伤模型进行比较，因为这些模型与炎症不同，周围神经损伤(轴突切断)后DRG基因表达的时间进程的分析也已部分完成。目前正在收集结果，以供出版。我们还创建了一个共享数据的网站，这将成为其他团体获取数据的公开网站的基础。

Summary: This project is in the beginning stages. At this point we have established several research methods and protocols, built the supporting infrastructure in terms of equipment, space in the lab, collaborative arrangements, hired and begun to train a team of scientists and support personnel to conduct the research project. Over the course of this year we have performed over 150 sequencing runs on the Illumina HiSeq 2000 and examined peripheral inflammation, peripheral nerve damage, models of arthritis, sequencing of genetically labeled neuronal populations and completed the trigeminal transcriptome in several species. We are in the process of writing multiple reports on these data. Some of the most interesting observations relate to the expression of the exact genes that mediate the actions of known analgesic drugs such as morphine, clonidine and sodium and calcium channel blockers. We can now assign which nociceptive neuronal population is controlled by which analgesic drug and, because we are examining both model species and human post-mortem tissue, we can generalize to the human and focus analgesic research on the most relevant known and novel molecular targets. The personnel now include a Ph.D. research fellow trained in biochemistry and bioinformatics, a predoctoral fellow trained in bioinformatics and computational biology, and a post-BAC IRTA with experience in RNA extraction who also will be trained in cDNA library construction and RT-PCR analysis. The laboratory has purchased two dedicated computer workstations for high-speed sequence alignment and analysis of the transcriptome libraries and constructed a third. We upgraded our Bioanalyzer for RNA quality assessment and utilize 96 and 384 well real-time PCR devices for independent verification of mRNA alterations seen with the RNA-seq methods. For sequencing we work with investigators at the National Institutes Sequencing Center (NISC) and mainly use the Illumina High-Seq 2000 platform for deep sequencing, generally in multiplex mode to analyze 6 independent samples/lane. We determined that polyA+ selection was superior to ribosomal RNA depletion for construction of the sequencing libraries. We contracted with the University of Pennsylvania to obtain canine tissue for the RNA studies. These tissues will be obtained from controls and animals with osteosarcoma euthanized because of inadequate pain control or treated with resiniferatoxin and tissues obtained at autopsy. The data obtained will allow us to test for genes activated by nociceptive input from naturally occurring bone cancer and modulated by treatment. Accrual in the canine study is nearly complete. The data will also be used for comparison to parallel studies in mouse, rat and human (although the exact models will be different). The transcriptome project is systematically investigating the first three steps in the pain pathway beginning with injured peripheral tissue or nerve, the dorsal root ganglion and the dorsal (sensory) spinal cord. The equivalent structures for the face and head are the trigeminal ganglia and the medulla (medullary dorsal horn). We have obtained and analyzed these two tissues in collaboration with Dr. Joel Kleinman of NIMH. These same tissues will be analyzed from rat, dog and monkey. Using the Bioanalyzer, we have analyzed total RNA extracted from the human medullary dorsal horn and trigeminal ganglia. After several technical improvements, analysis yielded excellent RNA integrity numbers. We have nearly completed the initial study on inflammation-induced genes in rats. This includes a systematic examination of the time course of gene regulation in inflamed peripheral tissue, DRG and dorsal horn. Comparisons will be made to several nerve injury models since these are distinct from the inflammation and the analysis of the time course of DRG gene expression following peripheral nerve injury (axotomy) has also been partially completed. The results are being assembled for publications. We have also created a web site for data sharing and this will form the basis for a publically accessible site for other groups to access the data.