Cytokine Receptor Expression Profiling

Expressions of cytokine receptors were semiquantitatively measured by RT-PCR as previously described [40 (link)]. Total RNA was extracted by the TRIzol™ Reagent (Thermo Fisher Scientific), and cDNA was synthesized by Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. RT-PCR was performed using the LightCycle 480^® real-time PCR system (Roche Diagnostic, Mannheim, Germany). Forty nanograms of cDNA, 0.4 μM primers, and LightCycle 480^® SYBR green I master mix (Roche Diagnostic) was included. The gene expression level was quantified by LightCycle 480^® Relative Quantification software (Roche Diagnostic). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was an internal control. Oligonucleotide primers are listed in Table 1.