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RNA levels in a cell are determined by the relative rates of RNA synthesis and decay. State-of-the-art transcriptional analyses only employ total cellular RNA. Therefore, changes in RNA levels cannot be attributed to RNA synthesis or decay, and temporal resolution is poor. Recently, it was reported that newly transcribed RNA can be biosynthetically labeled for 1-2 h using thiolated nucleosides, purified from total cellular RNA and subjected to microarray analysis. However, in order to study signaling events at molecular level, analysis of changes occurring within minutes is required. We developed an improved approach to separate total cellular RNA into newly transcribed and preexisting RNA following 10-15 min of metabolic labeling. Employing new computational tools for array normalization and half-life determination we simultaneously study short-term RNA synthesis and decay as well as their impact on cellular transcript levels. As an example we studied the response of fibroblasts to type I and II interferons (IFN). Analysis of RNA transcribed within 15-30 min at different times during the first three hours of interferon-receptor activation resulted in a &gt;10-fold increase in microarray sensitivity and provided a comprehensive profile of the kinetics of IFN-mediated changes in gene expression. We identify a previously undisclosed highly connected network of short-lived transcripts selectively down-regulated by IFNgamma in between 30 and 60 min after IFN treatment showing strong associations with cell cycle and apoptosis, indicating novel mechanisms by which IFNgamma affects these pathways.

High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay