title: Identification of a common reference gene pair for qPCR in human mesenchymal stromal cells from different tissue sources treated with VEGF.
abstract: 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.
authors.forename: Josefine
authors.initials: J
authors.lastname: Tratwal
authors.forename: Bjarke
authors.initials: B
authors.lastname: Follin
authors.forename: Annette
authors.initials: A
authors.lastname: Ekblond
authors.forename: Jens
authors.initials: J
authors.lastname: Kastrup
authors.affiliation: Cardiology Stem Cell Centre, The Heart Centre, Rigshospitalet, Copenhagen University Hospital, Juliane Maries Vej 20, dept, 9302, 2100 Copenhagen, Denmark. Mandana.Haack-Soerensen@regionh.dk.
authors.forename: Mandana
authors.initials: M
authors.lastname: Haack-Sørensen
mesh_terms: D000328:Adult; D001854:Bone Marrow Cells / Q000378:metabolism*; D005260:Female; D005796:Genes / Q000235:genetics*; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000378:metabolism*; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods; D035181:TATA-Box Binding Protein / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
chemical_list: D035181:TATA-Box Binding Protein; D042461:Vascular Endothelial Growth Factor A