Quantification of Inflammatory Cytokine Expression

Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer's instructions, and reverse transcription was performed using a Takara reverse transcription kit (Takara, Shiga, Japan). The expression of IL-6, TNF-α and IFN-β was quantified using SYBR Premix Ex Tap™, with GAPDH as an internal normalized reference. The specific sequences of the primers used were as previously described (3 (link),13 (link)). Quantitative polymerase chain reaction (qPCR) was performed under the following conditions: 95°C for 30 sec, followed by 45 cycles at 95°C for 5 sec, 60°C for 5 sec, 72°C for 5 sec and 65°C for 20 sec, using the LightCycler Real-time PCR system (Roche Diagnostics, Indianapolis, IN, USA) as previously described (14 (link)).