EF1alpha and RPL13a represent normalization genes suitable for RT-qPCR analysis of bone marrow derived mesenchymal stem cells.
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, EF1alpha, GAPDH, RPL13a, YWHAZ, UBC and HPRT1 were tested as possible housekeeping genes based on their expression level and variability. EF1alpha 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 EF1alpha, 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.
Curtis
Kevin M
KM
Geriatric Research, Education, and Clinical Center, Veterans Affairs Medical Center and The Geriatrics Institute, 1201 NW 16th Street, Miami, Florida 33125 USA.
Gomez
Lourdes A
LA
Rios
Carmen
C
Garbayo
Elisa
E
Raval
Ami P
AP
Perez-Pinzon
Miguel A
MA
Schiller
Paul C
PC
D048948:14-3-3 Proteins / Q000235:genetics; D000818:Animals; D001854:Bone Marrow Cells / Q000502:physiology*; D002545:Brain Ischemia / Q000235:genetics; D004815:Epidermal Growth Factor / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D018832:Molecular Chaperones / Q000235:genetics; D009363:Neoplasm Proteins / Q000235:genetics*; D058953:Neural Stem Cells / Q000502:physiology; D010100:Oxygen / Q000378:metabolism; D020648:Peptide Elongation Factor 1 / Q000235:genetics*; D051381:Rats; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012269:Ribosomal Proteins / Q000235:genetics*; D055815:Young Adult
D048948:14-3-3 Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D020648:Peptide Elongation Factor 1; C085488:RPL13 protein, human; D012269:Ribosomal Proteins; C490391:Ywhaz protein, rat; D016222:Fibroblast Growth Factor 2; D004815:Epidermal Growth Factor; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010100:Oxygen