Transcriptional Profiling of Macrophage Cytokines

Total RNA from macrophages was isolated using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Reverse transcription was carried out using 1.5 mcg total RNA and oligo(dT)₁₈ primers with RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) according to manufacturer's protocol. Real-time quantitative PCR was performed using qPCRmix-HS SYBR+LowROX Kit (Evrogen, Moscow, Russia) on the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). RT-PCR analysis was performed as described previously (Korneev et al., 2015 (link)). The following primers were used: IL-6, 5′-CTC TGC AAG AGA CTT CCA TCC, 5′-TTC TGC AAG TGC ATC ATC GT; TNF, 5′-TCT GTC TAC TGA ACT TCG GG, 5′-TTG GTG GTT TGC TAC GAC; IL-1β, 5′-TCA ACC AAC AAG TGA TAT TCT CCA T, 5′-ACT CCA CTT TGC TCT TGA CTT CT; RANTES, 5′-CCC TCA CCA TCA TCC TCA C, 5′-CCT TCG AGT GAC AAA CAC GA; IP-10, 5′-AAG TGC TGC CGT CAT TTT CT, 5′-GTG GCA ATG ATC TCA ACA CG; IRF3, 5′-AAC CGG AAA GAA GTG TTG CG, 5′-GCA CCC AGA TGT ACG AAG TC and β-actin, 5′-GAC CTC TAT GCC AAC ACA GT, 5′-AGA AAG GGT GTA AAA CGC AG.