Localization of Plasmodium lncRNAs via RNA FISH

RNA FISH was performed with slight modifications as described by Sierra-Miranda, 2012^{59 (link)} on mixed-stage asexual and gametocyte stage parasites. Antisense RNA probes for seven nuclear lncRNAs; -TARE4, −178, −13, −1494, −271, −4076 -ch9, -ch14 and two cytoplasmic lncRNAs; −267, −643, were labeled by in vitro transcription in the presence of fluorescein. RNA FISH was also performed using sense RNA probes as controls. Briefly, fixed and permeabilized parasites were incubated with RNA probes overnight at 37 °C. Parasites were washed with 2x SSC three times for 15 min each at 45 °C followed by one wash with 1x PBS for 5 min at room temperature. The slides were mounted in a Vectashield mounting medium with DAPI and visualized using the Olympus BX40 epifluorescence microscope. Images were treated with ImageJ. Pictures are representative of 15-20 positive parasites examined.