Global transcriptomic changes of Plasmodium falciparum asexual parasites and late gametocytes after treatment with MMV390048 or MMV642943. Plasmodium falciparum

Plasmodium falciparum asexual parasites and late gametocytes were treated for 24 or 48 h with MMV390048 or MMV642943 in order to understand the functional consequences of inhibiting lipid kinases Overall design: Agilent Plasmodium falciparum DNA microarrays were performed to analyze the global transcriptomic profiles of asexual parasites and late stage gametocytes following 24 or 48 h treatment with MMV390048 or MMV642943. NF54 ring-stage ( >95% synchronicity, 6 hours post merozoite invasion) asexual parasites and mixed stage IV and V gametocytes were treated with 10 X IC50 of MMV390048 or MMV642943 for 24 or 48 h. Thereafter, parasitised erythrocytes were washed and stored at -70°C. Gametocytes were isolated from erythrocytes using Nycoprep, washed and stored at -70°C. RNA was isolated using a a combination of TriZol reagent and Qiagen RNeasy kit. RNA from each of the 10 samples labelled with Cy5 was hybridised against Cy3 labelled reference pool consisting of untreated and treated mixed asexual parasites and gametocytes. Arrays were scanned using the Axon GenePix 4000B scanner. Images were quantified and data was normalized with GenePix Pro 5.1 software.

为探究脂质激酶（lipid kinases）抑制所产生的功能效应，本研究使用MMV390048与MMV642943对恶性疟原虫（Plasmodium falciparum）无性期寄生虫与晚期配子体分别处理24小时或48小时。
实验设计：采用安捷伦（Agilent）恶性疟原虫DNA微阵列（DNA microarrays），分析经MMV390048或MMV642943处理24或48小时后的无性期寄生虫与晚期配子体的全局转录组谱（global transcriptomic profiles）。
取NF54株环期（同步率>95%，裂殖子入侵后6小时）无性期寄生虫，以及混合IV、V期配子体，以10倍IC50浓度的MMV390048或MMV642943处理24或48小时。随后将感染的红细胞洗涤后于-70℃保存。配子体通过Nycoprep试剂从红细胞中分离，洗涤后于-70℃保存。
采用TriZol试剂（TriZol reagent）与Qiagen RNeasy试剂盒（Qiagen RNeasy kit）联用的方法提取总RNA。将10个均经Cy5标记的样品RNA，与由未处理及处理后的混合无性期寄生虫、配子体组成的Cy3标记参考混合池进行杂交。使用Axon GenePix 4000B扫描仪（Axon GenePix 4000B scanner）对微阵列进行扫描，通过GenePix Pro 5.1软件（GenePix Pro 5.1 software）完成图像定量与数据标准化。