Enterococcus faecalis OG1RF control vs. treatment with vancomycin

Transcriptional profiling to investigate the effect of drug treatment on the E. faecalis cells. For microarray analysis, E. faecalis OG1RF was grown in FMC medium supplemented with 10 mM glucose to an optical density at 600 nm (OD600) of 0.3 and the cultures were divided in 3 aliquots. One aliquot was collected by centrifugation and immediately frozen (untreated control cells). The other aliquots were treated for 30 or 60 min with 1.25 X the minimum inhibitory concentration (MIC) of vancomycin (10 μg ml-1). After an exposure time of 30 or 60 minutes, each of these cultures was also centrifuged and the pellets frozen. RNA was then isolated from each pellet for microarray analysis. This process was repeated 3 additional times, for a total of four replicates of each condition. RNA was extracted from four replicate samples of each condition of interest (control cells grown to OD600 = 0.3 in FMC medium supplemented with 10mM glucose, then treated with vancomycin for 30 or 60 minutes) and labeled with Cy3. For each replicate, labeled RNA was hybridized to slides along with Cy5-labeled reference RNA, extracted from E. faecalis OG1RF cells grown in BHI medium to mid-log.本研究采用转录谱分析（Transcriptional profiling）技术，探究药物处理对粪肠球菌（E. faecalis）细胞的影响。针对微阵列分析（microarray analysis）实验，将粪肠球菌OG1RF株接种于添加10 mM葡萄糖的FMC培养基（FMC medium）中培养，待其600 nm光密度（OD600）达到0.3时，将培养液均分为三份等分试样。其中一份等分试样经离心（centrifugation）收集后立即冷冻保存，作为未处理对照细胞。剩余两份等分试样分别用1.25倍最低抑菌浓度（minimum inhibitory concentration, MIC）的万古霉素（vancomycin，MIC值为10 μg·ml⁻¹）处理30分钟与60分钟。待暴露处理时长分别达到30分钟或60分钟后，将这两组培养液同样通过离心收集菌体沉淀并冷冻保存。随后从每份菌体沉淀中提取核糖核酸（RNA）用于微阵列分析。该实验流程共重复3次，最终每种处理条件均获得4次生物学重复。从各目标处理条件的4份重复样本中提取RNA，目标处理条件包括：在添加10 mM葡萄糖的FMC培养基中培养至OD600=0.3的对照细胞，以及经万古霉素处理30分钟或60分钟的细胞；所提取的RNA均采用Cy3进行荧光标记。对于每一份重复样本，将标记有Cy3的待测RNA与标记有Cy5的参照RNA共同杂交至芯片玻片；该参照RNA提取自在脑心浸液培养基（BHI medium）中培养至对数中期的粪肠球菌OG1RF细胞。