Quantifying STAT3 Expression in Polarized Macrophages

Total RNA was isolated from 1 × 10⁶ cytokine-polarized macrophages using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA), and quantified in a NanoDrop OneC spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA). Then, 1 µg of isolated RNA was used to synthesize cDNA for each sample using the RT2 First Strand Kit (Qiagen, Hilden, NRW, Germany) and following the manufacturer’s instructions. The following primers were used: for STAT3 amplification, 5′-GCTTTTGTCAGCGATGGAGT-3′ (forward), 5′-ATTTGTTGACGGGTCTGAAGTT-3′ (reverse) [126 (link),127 (link)], and for the GAPDH housekeeping control, 5′-CTTCACCACCATGGAGAAGGC-3′ (forward), 5′-GGCATGGACTGTGGTCATGAG-3′ (reverse) [128 (link)]. The PCR reaction mixture was performed in a final volume of 12.5 µL, which contained 2X SYBR^® Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA), 250 nM of each primer, and 25 ng of cDNA. The following thermal cycling was employed, an initial step at 95 °C for 10 min; followed by 50 denaturation cycles at 95 °C for 20 s, annealing at 59 °C for 20 s and extension at 72 °C for 20 s. The amplification was performed in a Rotor-Gene Q 5 PLEX HRM (Qiagen, Hilden, NRW, Germany) and was analyzed in the Rotor-Gene 114 Q Software 2.3 (Qiagen, Hilden, NRW, Germany). The 2^−ΔΔCt method was used to determine relative expression.