Selection of Stable Reference Genes for Gene Expression Studies in Activated and Non-Activated PBMCs Under Normoxic...

Immunotherapy has emerged as a key modality in cancer treatment, yet its effectiveness varies significantly among patients, often due to the metabolic stress imposed by the tumor microenvironment. Hypoxia, a major factor in the tumor microenvironment, results from the high metabolic rate of tumor cells and inadequate vascularization, impairing immune cells' function and potentially influencing gene expression profiles. Despite the widespread use of quantitative real-time PCR in immunological studies, to the best of our knowledge, data on reference gene stability in human peripheral blood mononuclear cells under hypoxic conditions is limited. In our study, we assessed the expression stability of commonly used reference genes (S18, HPRT, IPO8, RPL13A, SDHA, PPIA, and UBE2D2) in both non-stimulated and CD3/CD28-activated peripheral blood mononuclear cells cultured under normoxic, hypoxic (1% O₂), and chemically induced hypoxic conditions for 24 h. Analysis using four different algorithms-delta Ct, geNorm, NormFinder, and BestKeeper-identified RPL13A, S18, and SDHA as the most suitable reference genes for human peripheral blood mononuclear cells under hypoxic conditions. In contrast, IPO8 and PPIA were found to be the least suitable housekeeping genes. The study provides essential insights into the stability of reference genes in peripheral blood mononuclear cells under hypoxic conditions, a critical but understudied aspect of immunological research. Given the significant impact of hypoxia on T cell metabolism and function in the tumor microenvironment, selecting reliable reference genes is crucial for accurate gene expression analysis. Our findings will be valuable for future studies investigating hypoxia-driven metabolic reprogramming in immune cells, ultimately contributing to a better understanding of T cell responses in cancer immunotherapy.