Quantitative RT-PCR Analysis of Gene Expression

For gene expression analysis, total RNA from BMDMs was extracted using the TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction. RNA was reverse transcribed (1 mg of total RNA) using Illustra Ready-To-Go RT-PCR Beads (GE Healthcare, Chicago, IL) as previously shown (Guimaraes et al., 2019 (link)). The PCR reaction was performed with QuantStudio3 real-time PCR instrument (Applied Biosystems) as previously described (Guimaraes et al., 2019 (link)). The primers were used to amplify a specific fragment corresponding to specific gene target: β-actin, forward, 5’-GGC TGT ATT CCC CTC CAT CG-3’, reverse, 5’-CCA GTT GGT AAC AAT GCC ATG T-3’; Galectin-3, forward, 5’-CAG GAA AAT GGC AGA CAG CTT-3′, reverse, 5’- CCC ATG CAC CCG GAT ATC-3′; IFN-β, forward, 5’-GCC TTT GCC ATC CAA GAG ATG C-3’, reverse, 5’-ACA CTG TCT GCT GGT GGA GTT C-3’; GBP2, forward, 5’-CTG CAC TAT GTG ACG GAG CTA-3’, reverse, 5’-CGG AAT CGT CTA CCC CAC TC-3’; GBP3, forward, 5’-CTG ACA GTA AAT CTG GAA GCC AT-3’, reverse, 5’-CCG TCC TGC AAG ACG ATT CA-3’; GBP4, forward, 5’-GGA GAA GCT AAC GAA GGA ACA A-3’, reverse, 5’-TTC CAC AAG GGA ATC ACC ATT TT-3’; GBP5, forward, 5’-CTG AAC TCA GAT TTT GTG CAG GA-3’, reverse, 5’-CAT CGA CAT AAG TCA GCA CCA G-3’. The results are presented as relative expression units after normalization to the β-actin gene. The experiments were conducted in triplicate.