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 CXCR4, CCR1, XIAP, CCR3, NOTCH1, ZBTB16, PAK1, CASP9, TNFAIP3, BCL2L11 and TSC22D3. 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.