RNA profiling is an important technology that measures RNA levels in cells and tissues, and that has beenused to define pathways in mammalian organ development. Because the central focus of the SysCODEConsortium 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 issuesencountered with conventional microarray platforms in the analysis of low abundance transcripts. ThePMAGE method combines the frequency distribution technique pioneered by SAGE (serial analysis of geneexpression) with the high throughput Polony DNA sequencing technique to produce histograms, whichaccurately profile the RNAs in tissues or cells. That is, a single 14 base pair tag is produced from a singleRNA 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 SAGElibraries, 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 encodingtranscription factors and other regulatory molecules that may be important to a molecular understanding oforganogenesis. The ability to comprehensively define levels of transcription factors should therefore providenovel insights into organ development. We propose to create, under the aegis of this P30 Core grant, aPMAGE Technology Development Core facility that will enable Consortium investigators to determineexpression profiles from total RNA. We expect to produce about 50 PMAGE RNA profiles in the first yearand 250 RNA profiles during the grant period. These RNA profiles will provide unique insights into themechanisms by which the tooth germ, pancreatic islet and heart valve form. In particular we will define thecomplete catalogue of transcription factors that are required for the development of these tissues.
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