Plasmodium falciparum whole-genome real-time transcription and decay. Plasmodium falciparum

To date, total mRNA analysis throughout intraerythrocytic development of the malaria parasite, Plasmodium falciparum, has only revealed abundance profiles of each gene at a given time. Here, we reveal that biosynthetic pyrimidine labeling allows for the calculation of real-time, whole-genome analysis of transcription and stability throughout asexual development. In turn, we are now able to determine transcriptional and decay rates at any given time during development of specific genes or subsets of genes which will drive new investigations on both transcriptional and post-transcriptional regulatory dynaimc and networks. Overall design: A single timecourse experiment in which 48 individual cultures were highly synchronized and, at a given hour post invasion (0-47), were treated with 40um 4-TU for 10min followed immediately by Trizol total RNA extraction. Total RNA from each timepoint was then biotinylated via a thiol-group on any transcript that incorporated a modified UTP. Streptavidin modified transcripts were then separated from total RNA by Streptavidin magentic beads resulting in a Labeled sample. Any mRNA that was not bound to the beads resulted in an Unlabeled sample. A fraction of the total RNA was untreated and resulted in a Total sample. Each hour, three samples were analyzed by microarray, Total, Unlabeled, and Labeled, resulting in 144 individual microarrays (48 for each sample type).