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 该联盟将在发育中的牙胚、胰岛和心脏瓣膜中定义这样的通路， 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，可以避免潜在的问题， 在低丰度转录物的分析中遇到的常规微阵列平台。的 PMAGE方法结合了SAGE（基因序列分析）开创的频率分布技术， 表达）与高通量Polony DNA测序技术产生直方图， 准确地分析组织或细胞中的RNA。也就是说，单个14个碱基对标签由单个14个碱基对标签产生。 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。 通过这种方法获得的RNA图谱包含的标签比大多数SAGE多约30倍。 因此能够测量以每个细胞少于0.3个RNA拷贝表达的RNA水平。 因此，这种增强的灵敏度涵盖了潜在的低丰度RNA，例如编码 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核心补助金的支持下， PMAGE技术开发核心设施，使财团调查人员能够确定 总RNA的表达谱。我们预计在第一年将产生大约50个PMAGE RNA图谱 和250个RNA图谱。这些RNA图谱将为我们提供独特的见解， 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。 履约地点（组织、城市、州） 哈佛医学院遗传学系，波士顿，MA 02115 PHS 398（2004年9月修订版）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