Quantstudio 5 fast real time pcr system

Total RNA was extracted from treated and untreated THP1 cells using RNeasy Mini Kit (Qiagen, Valencia. CA, USA). One microgram of total RNA was used for cDNA conversion using high capacity cDNA reverse transcription kit (Applied Biosystems, Foster city, CA, USA). Real time polymerase chain reaction was performed using 50ng of cDNA, gene specific TaqMan Gene Expression Assays containing gene-specific primers and TaqMan MGB probe (6-FAM dye-labeled) and TaqMan® Gene Expression Master Mix (Applied Biosystems, Foster city, CA, USA) in a QuantStudio 5Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The Ct values of CCL4 and SAPK/JNK were normalized with corresponding GAPDH Ct values and the expression level of CCL4 in treated samples relative to control samples were calculated with -2ΔΔCt-method. Relative mRNA expression was expressed as fold expression over average of control gene expression. The expression level in control treatment was assumed to be 1.