Open AccessWhat is beyond a q RT ‐ PCR study on mesenchymal stem cell differentiation properties: how to choose the most reliable housekeeping genes
Author(s) -
Ragni Enrico,
Viganò Mariele,
Rebulla Paolo,
Giordano Rosaria,
Lazzari Lorenza
Publication year - 2013
Publication title -
journal of cellular and molecular medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.44
H-Index - 130
eISSN - 1582-4934
pISSN - 1582-1838
DOI - 10.1111/j.1582-4934.2012.01660.x
Subject(s) - housekeeping gene , mesenchymal stem cell , biology , regenerative medicine , cellular differentiation , stem cell , gene expression , gene expression profiling , adipogenesis , gene , computational biology , chondrogenesis , cell , microbiology and biotechnology , bioinformatics , genetics
Abstract 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 q RT ‐ PCR to obtain validated and comparable results. By our knowledge, an exhaustive validation study of HG s comparing MSC s 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 , RPL 13A , RPLP 0 , TBP , UBC , YWHAZ and 18S r RNA ) to assess their suitability as HG s in MSC s during adipogenic, osteogenic and chondrogenic differentiation. We demonstrated that many of the most popular HG s including 18S r RNA , 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 HG s choice on the quantification of cell differentiation markers, thus interfering with a reliable comparison of the lineage potential properties among various MSC s. 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.