Nascent mRNA profiling of LPS-stimulated mouse macrophages

To identify transcriptionally regulated genes in primary mouse macrophages stimulated with LPS with high sensitivity, we isolated nascent RNA following metabolic labelling with 4-thiouridine during the last 35 min before cell harvest, as recently described (Dolken et al. 2008 RNA 14:1959-72). Microarray analyses of nascent RNA identified substantially more probe sets as up-regulated after 45 min of LPS stimulation than parallel analyses of total cellular RNA. In contrast, 4.5 h after stimulation, up-regulated genes in total and nascent RNA largely overlapped. This approach therefore allowed a much more sensitive detection of early changes in transcription, and the respective genes are likely to be direct targets of LPS-regulated transcription factors. Keywords: Effect of LPS stimulation; comparison of changes in expression of total versus nascent RNA; timecourse Two completely independent experiments were performed. Macrophages were stimulated with 100 ng/ml LPS for 45 or 270 minutes. Thiouridine pulsing was done in last 35 minutes before harvest. Total RNA was isolated. Nascent RNA was subsequently purified.