Selection and evaluation of reliable reference genes in PMA-differentiated THP-1 macrophages following LPS stimulation.

Macrophages are innate immune cells differentiated from peripheral blood monocytes and play a pivotal role in immune responses against various antigens. However, the number of primary monocytes available for functional studies is limited due to their poor proliferative property. The human monocytic cell line, THP-1 is widely used as a simplified model for studying primary human macrophages because it can be differentiated into macrophage-like THP-1 cells (THP-1/M) by phorbol 12-myristate 13-acetate (PMA). In this study, expression of 12 candidate reference genes was measured by RT-qPCR in PMA-differentiated THP-1/M subjected to short-term lipopolysaccharide (LPS) stimulation (3 h; M3) or long-term LPS stimulation (24 h; M24), the latter following a 2-day resting phase. Expression stability was assessed using four algorithms: geNorm, NormFinder, BestKeeper, and RankAggreg. We found that the RPLP0/HPRT1 and GAPDH/PPIB pairs were the most stably expressed genes under the M3 and M24 conditions, respectively. In contrast, B2M was considered as the least stable gene under both conditions. Furthermore, analysis of TNF and IL1B expression revealed that normalization using other candidate reference genes resulted in differences in relative expression levels and/or expression variations compared to normalization using the selected optimal genes under both conditions. These results indicate that, even at the same LPS concentration, the most reliable reference genes in THP-1/M cells may differ depending on the protocol, including the PMA-free resting period and LPS duration; therefore, reliable reference genes should be determined for gene expression analysis in every specific protocol for THP-1 differentiation into THP-1/M cells and their subsequent stimulation.