Double staining of Plasmodium falciparum nucleic acids with hydroethidine and thiazole orange for cell cycle stage analysis by flow cytometry.

Abstract

Microarray analyses of stage-specific gene expression of Plasmodium falciparum require purification of RNAs from highly synchronized cultures. To date, no reliable method to control the quality of synchronization of P. falciparum cultures is available. A double-staining method using hydroethidine and thiazole orange for nucleic acid staining was carried out to compare by flow cytometric analysis the nucleic acid labeling of synchronized P. falciparum in cultures at different time points of the 48-h intraerythrocytic cycle. With this method, we determined the quality of culture synchronization in schizont and ring stages. Nucleic acid analysis, based on thiazole orange fluorescence, clearly showed that low levels of schizonts in ring cultures results in a high contamination of ring nucleic acids by schizonts. Conversely, nucleic acids from trophozoite or schizont cultures containing ring stages did not present a significant contamination by ring nucleic acids. The results demonstrated a very low nucleic acid content in the ring stage when compared with the high nucleic acid content of schizont-stage parasites. The rapid and reliable flow cytometric strategy using hydroethidine- and thiazole orange-stained parasite nucleic acids allows monitoring of the purity of the preparation, thus greatly improving the quality assessment of parasite cultures, a critical step to study gene expression patterns.