Capturing the dynamic RNA landscape of the malaria parasite P. 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 establish a new methodology in Plasmodium falciparum that enables biosynthetic labeleing and capture of sub-population mRNA. As a proof of principle for this novel method, we examine the mRNA dynamics of early gametocyte commitment. Plasmodium falciparum strains 3D7, E5, and F12 were highly synchronized and, at 36 hours post invasion, incubated with 40um 4-TU for 12 hours 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 thiol-modified UTP. Biotinylated transcripts were then separated from total RNA by Streptavidin magnetic beads resulting in a Labeled sample. Any mRNA that was not bound to the beads resulted in an Unlabeled sample. Every 12 hours, two samples were analyzed by Agilent P. falciparum DNA microarray, Unlabeled and Labeled, resulting in 48 individual DNA microarrays (4 for each sample type).