Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) for in vitro Polarization

In this study, we used LPS and IL-4 for in vitro microglial activation and polarization. On Day 11, LPS (10 μg/ml) or IL-4 (20 ng/ml) were added to the iMG cells and incubated for 24 h at 37°C with 5% CO₂. The next day, cell morphology was monitored and captured for each group. The total RNA was isolated using Trizol according to the manufacturer's instruction and subjected to cDNA synthesis using SuperScript™ IV VILO™ Master Mix. With the following primers, for M1 (TNF-α) and M2 (CD206) gene-specific primers, qRT-PCR was performed using PowerUp SYBR Green PCRmix. The relative expression was normalized to the housekeeping control gene β-actin. Fold changes in mRNA levels between stimulated and non-stimulated cells were calculated using the 2^−(ΔΔCT) method.

The sequences of primers were as follows:

β-actin F 5′-GATGCAGAAGGAGATCACTGC-3′

β-actin R 5′-ATACTCCTGCTTGCTGATCCA-3′

TNF-α F 5′-CCCAGGCAGTCAGATCATCTTCT-3′

TNF-α R 5′-ATGAGGTACAGGCCCTCTGAT-3′

CD206 F 5′-GTCATTCCGGGTGCTGTTCTCC-3′

CD206 R 5′-TTCGGCATCCTGGTTGCAAG-3′