Current methods to analyze gene expression measure steady-state levels of mRNA. To specifically analyze mRNA transcription, we have developed a technique that can be applied in vivo in intact cells and animals. Our method makes use of the cellular pyrimidine salvage pathway and is based on affinity-chromatographic isolation of thiolated mRNA. When combined with data on mRNA steady-state levels, this method is able to assess the relative contributions of mRNA synthesis and degradation/stabilization. It overcomes limitations associated with currently available methods such as mechanistic intervention that disrupts cellular physiology, or the inability to apply the techniques in vivo. Our method was first tested in serum response of cultured fibroblast cells and then applied to the study of renal ischemia reperfusion injury, demonstrating its applicability for whole organs in vivo. Combined with data on mRNA steady-state levels, this method provided a detailed analysis of regulatory mechanisms of mRNA expression and the relative contributions of RNA synthesis and turnover within distinct pathways, and identification of genes expressed at low abundance at the transcriptional level.newly transcribed RNA ͉ posttranscriptional regulation ͉ RNA degradation/stabilization I n response to environmental changes, cells alter their gene expression program. Eukaryotic gene expression is a complex process that is fine-tuned at different levels, such as transcription, turnover, and translation of mRNAs. Microarray-based measurements of steady-state mRNA levels do not distinguish between different regulatory processes. Both transcriptional and posttranscriptional regulation, however, can profoundly influence steady-state mRNA levels (1-4). To evaluate de novo transcription and the relative contributions of mRNA transcription and turnover, nuclear transcription run-on and its modifications have long been the methods of choice (4-6). These methods, however, are either inherently cell-invasive and/or are not compatible with the assay formats currently used to analyze global gene expression. To avoid mechanistic intervention that disrupts cellular physiology for example by cell permeabilization, we modified a method in which we take advantage of the cellular pyrimidine salvage pathway. Pyrimidine ribonucleotides may be de novo synthesized from simple molecules by energetically expensive multistep pathways. Alternatively, ribonucleotide pools are sustained by recycling of the basic components derived from their catabolism during normal RNA turnover, the socalled salvage pathway, a mechanism that is energetically less expensive. By this more favorable pathway, it is possible to label and selectively enrich newly transcribed (nascent) RNA molecules (NT-RNA; refs. 7-9) in vivo by incorporation of exogenous 4-thiouridine (s4U) and subsequent RNA purification by affinity chromatography. The RNA can then be further processed by standard protocols for hybridization to oligonucleotide microarrays. We refer to our method with the acronym NIAC-...

MicroRNAs (miRNAs) constitute a recently discovered class of small non-coding RNAs that regulate expression of target genes either by decreasing the stability of the target mRNA or by translational inhibition. They are involved in diverse processes, including cellular differentiation, proliferation and apoptosis. Recent evidence also suggests their importance for cancerogenesis. By far the most important model systems in cancer research are mammalian organisms. Thus, we decided to compile comprehensive information on mammalian miRNAs, their origin and regulated target genes in an exhaustive, curated database called Argonaute (). Argonaute collects latest information from both literature and other databases. In contrast to current databases on miRNAs like miRBase::Sequences, NONCODE or RNAdb, Argonaute hosts additional information on the origin of an miRNA, i.e. in which host gene it is encoded, its expression in different tissues and its known or proposed function, its potential target genes including Gene Ontology annotation, as well as miRNA families and proteins known to be involved in miRNA processing. Additionally, target genes are linked to an information retrieval system that provides comprehensive information from sequence databases and a simultaneous search of MEDLINE with all synonyms of a given gene. The web interface allows the user to get information for a single or multiple miRNAs, either selected or uploaded through a text file. Argonaute currently has information on 839 miRNAs from human, mouse and rat.