These authors contributed equally to this work.
What is beyond a qRT-PCR study on mesenchymal stem cell differentiation properties: how to choose the most reliable housekeeping genes
Article first published online: 11 JAN 2013
© 2012 The Authors Journal of Cellular and Molecular Medicine Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Journal of Cellular and Molecular Medicine
Volume 17, Issue 1, pages 168–180, January 2013
How to Cite
Ragni, E., Viganò, M., Rebulla, P., Giordano, R. and Lazzari, L. (2013), What is beyond a qRT-PCR study on mesenchymal stem cell differentiation properties: how to choose the most reliable housekeeping genes. Journal of Cellular and Molecular Medicine, 17: 168–180. doi: 10.1111/j.1582-4934.2012.01660.x
- Issue published online: 29 JAN 2013
- Article first published online: 11 JAN 2013
- Manuscript Accepted: 10 OCT 2012
- Manuscript Received: 22 MAY 2012
- regenerative medicine;
- mesenchymal stem cells;
- differentiation potency;
- housekeeping genes
In the last years, mesenchymal stem cells (MSCs) have been identified as an attractive cell population in regenerative medicine. In view of future therapeutic applications, the study of specific differentiation-related gene expression is a pivotal prerequisite to define the most appropriate MSC source for clinical translation. In this context, it is crucial to use stable housekeeping genes (HGs) for normalization of qRT-PCR to obtain validated and comparable results. By our knowledge, an exhaustive validation study of HGs comparing MSCs from different sources under various differentiation conditions is still missing. In this pivotal study, we compared the expression levels of 12 genes (ACTB, Β2M, EF1alpha, GAPDH, GUSB, PPIA, RPL13A, RPLP0, TBP, UBC, YWHAZ and 18S rRNA) to assess their suitability as HGs in MSCs during adipogenic, osteogenic and chondrogenic differentiation. We demonstrated that many of the most popular HGs including 18S rRNA, B2M and ACTB were inadequate for normalization, whereas TBP/YWHAZ/GUSB were frequently identified among the best performers. Moreover, we showed the dramatic effects of suboptimal HGs choice on the quantification of cell differentiation markers, thus interfering with a reliable comparison of the lineage potential properties among various MSCs. Thus, in the emerging field of regenerative medicine, the identification of the most appropriate MSC source and cell line is so crucial for the treatment of patients that being inaccurate in the first step of the stem cell characterization can bring important consequences for the patients and for the promising potential of stem cell therapy.