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

Abstract

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.