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Background  RT-qPCR analysis is a widely used method for the analysis of mRNA expression throughout the field of mesenchymal stromal cell (MSC) research. Comparison between MSC studies, both  in vitro  and  in vivo , are challenging due to the varied methods of RT-qPCR data normalization and analysis. Therefore, this study focuses on putative housekeeping genes for the normalization of RT-qPCR data between heterogeneous commercially available human MSC, compared with more homogeneous populations of MSC such as MIAMI and RS-1 cells.    Results  Eight genes including;  ACTB, B2M, EF1α, GAPDH, RPL13a, YWHAZ, UBC   and  HPRT1   were tested as possible housekeeping genes based on their expression level and variability.  EF1α  and  RPL13a  were validated for RT-qPCR analysis of MIAMI cells during expansion in varied oxygen tensions, endothelial differentiation, neural precursor enrichment, and during the comparison with RS-1 cells and commercially available MSC.  RPL13a  and  YWHAZ  were validated as normalization genes for the cross-species analysis of MIAMI cells in an animal model of focal ischemia.  GAPDH , which is one of the most common housekeeping genes used for the normalization of RT-qPCR data in the field of MSC research, was found to have the highest variability and deemed not suitable for normalization of RT-qPCR data.    Conclusions  In order to make comparisons between heterogeneous MSC populations, as well as adult stem cell like MSC which are used in different laboratories throughout the world, it is important to have a standardized, reproducible set of housekeeping genes for RT-qPCR analysis. In this study we demonstrate that  EF1α ,  RPL13a  and  YWHAZ  are suitable genes for the RT-qPCR analysis and comparison of several sources of human MSC during  in vitro  characterization and differentiation as well as in an  ex vivo  animal model of global cerebral ischemia. This will allow for the comparative RT-qPCR analysis of multiple MSC populations with the goal of future use in animal models of disease as well as tissue repair.

EF1α and RPL13a represent normalization genes suitable for RT-qPCR analysis of bone marrow derived mesenchymal stem cells