Expression profiling of the three clonotypic lineages of Toxoplasma gondii. Toxoplasma gondii

Expression profiling of the three clonotypic lineages dominating T. gondii populations in North America and Europe provides a first comprehensive view of the parasite transcriptome. Overall design: Prugniaud, RH, and VEG strain parasites were cultured in human foreskin fibroblast (HFF) cells as previously described (Roos, Donald et al. 1994). Prior to host cell rupture, cells were scraped from the flask and spun at 300g for 9 min. The resultant pellet was lysed with Buffer RLT from the Qiagen RNeasy Mini Kit (Valencia, CA) and RNA was extracted according to the manufacturer’s instructions. Labeled cRNA was created using the One-Cycle Labeling protocol in the Affymetrix GeneChip® IVT Labeling Kit (Santa Clara, CA). Hybridization, washing, and scanning of arrays was performed using standard Affymetrix instrumentation and protocols for 11 micron, 169 format arrays. Biological triplicates were generated for each strain and expression values computed using the RMA implementation (default parameters) in the affy package from Bioconductor (Gentleman, Carey et al. 2004).

对北美和欧洲弓形虫（T. gondii）种群中占主导地位的三种克隆型谱系开展表达谱分析，首次全面呈现了该寄生虫的转录组全貌。
总体实验设计：按照此前报道的方法（Roos, Donald等，1994），将Prugniaud、RH及VEG株弓形虫接种于人包皮成纤维细胞（human foreskin fibroblast, HFF）中进行培养。在宿主细胞破裂前，将细胞从培养瓶中刮取，并以300g相对离心力离心9分钟。收集得到的沉淀使用Qiagen RNeasy Mini试剂盒（加利福尼亚州瓦伦西亚）中的Buffer RLT进行裂解，并按照试剂盒说明书提取RNA。采用Affymetrix GeneChip® IVT标记试剂盒（加利福尼亚州圣克拉拉）的单循环标记流程制备标记后的cRNA。针对11微米间距、169格式的基因芯片，采用标准的Affymetrix仪器设备及实验流程完成杂交、洗涤与扫描操作。每个菌株均设置3次生物学重复，表达值通过Bioconductor的affy软件包中的RMA算法（默认参数）计算得到（Gentleman, Carey等，2004）。