Cytokine Gene Expression Analysis

cDNA was synthesized from mRNA with poly(dT) primers and Superscript II reverse transcriptase assay (Life Technologies, Rockville, MD, USA). Quantitative real-time PCR (qPCR) was performed with Rotor-Gene Q 5plex HRM (Qiagen) in 36-well Gene Discs using the QuantiFast SYBR Green RT-PCR Kit (Qiagen) following the manufacturer’s instructions. The PCR protocol is described in detail in the Supplementary Data.

Gene expression variations of TNFα, CCL2/MCP-1, CCL3/MIP-1 α, TGFβ, and IL10 were evaluated in terms of fold induction with respect to the housekeeping gene hypoxanthine phosphoribosyltransferase-1 (HPRT-1) by the 2-∆∆CT method. Experiments were conducted in triplicate. The sequence of primers used in this work is described in the Supplementary Data.