SysCODE: PMAGE Technology Development (10 of 10)

用于定义哺乳动物器官发育的途径。因为SysCODE的中心焦点是 该联盟将在发育中的牙胚、胰岛和心脏瓣膜中定义这种途径， 该U54联盟的项目2、3和4将使用RNA分析来定义开发过程中的RNA水平。 我们最近开发了一种新的RNA分析程序，PMAGE，它可以绕过潜在的问题 在分析低丰度转录本时遇到常规微阵列平台。这个 PMAGE方法结合了SAGE(基因序列分析)首创的频率分布技术 表达)使用高通量Polony DNA测序技术来产生直方图，这 准确地描述组织或细胞中的RNA。也就是说，从单个14个碱基对标签产生单个 RNA分子，标签存储在PMAGE文库中，每个文库约有200万个标签测序。 用这种方法得到的RNA图谱包含的标签比大多数SAGE多约30倍 文库，因此能够测量每个细胞低于0.3个RNA拷贝的RNA表达水平。 因此，这种增强的敏感性包含了潜在的低丰度RNA，如编码的RNA 转录因子和其他调节分子，可能是重要的分子理解 器官发生。因此，全面定义转录因子水平的能力应该提供 对器官发育的新见解。我们建议，在P30核心赠款的支持下，创建一个 PMAGE技术开发核心设施，将使财团调查人员能够确定 总RNA的表达谱。我们预计在第一年生产约50个PMAGE RNA图谱 以及在授予期间的250个RNA图谱。这些RNA图谱将提供对 牙胚、胰岛和心脏瓣膜形成的机制。特别是，我们将定义 这些组织发育所需的转录因子的完整目录。 演出现场(S)(组织、市、州) 马萨诸塞州波士顿哈佛医学院遗传学系，邮编：02115 小灵通398(09/04版)第2页表格第2页

used to define pathways in mammalian organ development. Because the central focus of the SysCODE Consortium is to define such pathways in the developing tooth germ, pancreatic islet and heart valve, Projects 2, 3 and 4 of this U54 Consortium will use RNA profiling to define RNA levels during development. We have recently developed a novel RNA profiling procedure, PMAGE, that circumvents potential issues encountered with conventional microarray platforms in the analysis of low abundance transcripts. The PMAGE method combines the frequency distribution technique pioneered by SAGE (serial analysis of gene expression) with the high throughput Polony DNA sequencing technique to produce histograms, which accurately profile the RNAs in tissues or cells. That is, a single 14 base pair tag is produced from a single RNA molecule, the tags are stored in PMAGE libraries, and about 2 million tags are sequenced per library. The RNA profiles obtained by this method contain about 30-fold more tags than the majority of SAGE libraries, and hence are able to measure the level of RNAs expressed at less than 0.3 RNA copies per cell. This enhanced sensitivity thus encompasses potential low abundance RNAs, such as those encoding transcription factors and other regulatory molecules that may be important to a molecular understanding of organogenesis. The ability to comprehensively define levels of transcription factors should therefore provide novel insights into organ development. We propose to create, under the aegis of this P30 Core grant, a PMAGE Technology Development Core facility that will enable Consortium investigators to determine expression profiles from total RNA. We expect to produce about 50 PMAGE RNA profiles in the first year and 250 RNA profiles during the grant period. These RNA profiles will provide unique insights into the mechanisms by which the tooth germ, pancreatic islet and heart valve form. In particular we will define the complete catalogue of transcription factors that are required for the development of these tissues. PERFORMANCE SITE(S) (organization, city, state) Dept of Genetics, Harvard Medical School, Boston, MA 02115 PHS 398 (Rev. 09/04) Page 2 Form Page 2