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Background  Human mesenchymal stromal cells from the bone marrow (BMSCs) are widely used as experimental regenerative treatment of ischemic heart disease, and the first clinical trials using adipose-derived stromal cells (ASCs) are currently being conducted. Regenerative mechanisms of BMSCs and ASCs are manifold and  in vitro  pretreatment of the cells with growth factors has been applied to potentially enhance these properties. When characterizing the transcriptional activity of these cellular mechanisms  in vitro  it is important to consider the effect of the growth factor treatment on reference genes (RGs) for the normalization of qPCR data.    Results  BMSCs and ASCs were stimulated with vascular endothelial growth factor A-165 (VEGF) for one week, and compared with un-stimulated cells from the same donor. The stability of nine RGs through VEGF treatment as well as the donor variation was assessed using the GenEx software with the subprograms geNorm and Normfinder.  The procedure of stepwise elimination was validated by poor performance of eliminated RGs in a normalization experiment using  vWF  as target gene. Normfinder found the TATA box binding protein  (TBP)  to be the most stable single RG for both BMSCs and ASCs. The optimal number of RGs for ASCs was two, and the lowest variance for  vWF  normalization was found using  TBP  and  YWHAZ . For BMSCs, the optimal number of RGs was four, while the two-RG combination producing the most similar results was  TBP  and  YWHAZ .    Conclusions  A common reference gene,  TBP , was found to be the most stable standalone gene, while  TBP  and  YWHAZ  were found to be the best two-RG combination for qPCR analyses for both BMSCs and ASCs through the VEGF stimulation. The presented stepwise elimination procedure was validated, while we found the final normalization experiment to be essential.

Identification of a common reference gene pair for qPCR in human mesenchymal stromal cells from different tissue sources treated with VEGF