qPCR in human eosinophils after glucocorticoid exposure

Eosinophils were isolated from the peripheral blood of each of five human subjects. Purified eosinophils were then incubated for 4 hours, followed by exposure to dexamethasone (0.5 mcM) or vehicle (culture medium) for an additional 30, 60, or 120 minutes. RNA was purified from each sample and reverse-transcription quantitative real-time PCR (qPCR) was performed on the QuantStudio6 Flex System with the TaqMan Fast Universal PCR Master Mix and FAM-labeled TaqMan gene expression assay sets for human <i>CXCR4</i>, <i>CCR1</i>, <i>XIAP</i>, <i>CCR3</i>, <i>NOTCH1</i>, <i>ZBTB16</i>, <i>PAK1</i>, <i>CASP9</i>, <i>TNFAIP3</i>, <i>BCL2L11</i> and <i>TSC22D3</i>. The gene encoding 18S rRNA was used as an endogenous control. The mean Ct values for each subject, under each condition (dexamethasone or medium), at each time point, were calculated as the mean of two technical replicates, and are presented in the Excel file, with one tab for each gene.

从5名人类受试者的外周血中分离嗜酸性粒细胞（Eosinophils）。将纯化后的嗜酸性粒细胞孵育4小时，随后分别用0.5微摩尔的地塞米松（dexamethasone）或溶剂（培养基）处理，处理时长分别为30、60或120分钟。从每份样本中纯化RNA，随后在QuantStudio6 Flex系统上采用TaqMan快速通用PCR预混液与FAM标记的TaqMan基因表达检测试剂盒，针对人类CXCR4、CCR1、XIAP、CCR3、NOTCH1、ZBTB16、PAK1、CASP9、TNFAIP3、BCL2L11及TSC22D3基因进行逆转录定量实时PCR（qPCR）。以编码18S核糖体RNA（18S rRNA）的基因作为内参。针对每名受试者、每种处理条件（地塞米松或培养基）及每个时间点，其平均Ct值为两次技术重复的均值；相关数据已整理至Excel文件中，每个基因对应一个工作表。