Quantifying Cytokine-Induced Gene Expression

Following cell synchronization for 24 h, cells were left unstimulated or were stimulated for 12, 24 or 48 h with 50 ng/mL TNF-α. Total RNA was extracted using RNeasy Mini RNA isolation kits (Qiagen, Valencia, Spain) in accordance with the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized using a High-Capacity cDNA Reverse transcription Kit (Applied Biosystems, Waltham, MA, USA) per the manufacturer’s protocol. CCL11, CCR3, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers were purchased from Qiagen. Quantitative PCR was performed using SYBR Green quantitative PCR master mix (Qiagen) on a C1000 thermal cycler (Bio Rad, Hercules, CA, USA) as described previously^{5 (link)}. The efficiency of the primer assays was guaranteed by the manufacturer to be > 90%. Each reaction was measured in triplicate, and the data were normalized to the expression levels of the housekeeping gene GAPDH. The ratio of each mRNA relative to the GAPDH mRNA was calculated using the ΔΔ threshold cycle method.