Evaluation of eight reference genes for quantitative polymerase chain reaction analysis in human T lymphocytes co‑cultured with mesenchymal stem cells.

Accurate gene expression analysis relies on the selection of a stable reference gene, as unstable reference genes can alter experimental results and conclusions. It is widely‑accepted that reference genes exhibit different expression levels in different types of tissues and cells. Therefore, it is essential to screen for stably‑expressed reference genes in the cells and tissues used for experimental analysis prior to performing reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). In the present study, eight reference genes were screened for their suitability for RT‑qPCR in five T lymphocytes co‑cultured with mesenchymal stem cells from different sources. Using NormFinder, geNorm, and BestKeeper algorithms consistently demonstrated that RPL13A and B2M were the optimal reference genes for the normalization of RT‑qPCR data obtained from T lymphocytes, whereas glyceraldehyde 3‑phosphate dehydrogenase was not a suitable reference gene due to its extensive variability in expression. These findings highlight the importance of evaluating reference genes for RT‑qPCR.