GAPDH, beta-actin and beta2-microglobulin, as three common reference genes, are not reliable for gene expression studies in equine adipose- and marrow-derived mesenchymal stem cells

• Published in: Journal of Animal Science and Technology Vol. 57; no. 5
• Main Authors: Nazari, F., Ferdowsi University of Mashhad, Mashhad, Iran, Parham, A., Ferdowsi University of Mashhad, Mashhad, Iran, Maleki, A.F., Ferdowsi University of Mashhad, Mashhad, Iran
• Format: Journal Article
• Language: English
• Published: 01.05.2015
• Subjects: CELLS; CELLULE; CELULAS; GAPDH,beta-actin,B2M,Reference gene,Mesenchymal stem cells,Equine; GENE; GENES
• ISSN: 1598-9429

Background: Quantitative real time reverse transcription PCR (qRT-PCR) is one of the most important techniques for gene-expression analysis in molecular based studies. Selecting a proper internal control gene for normalizing data is a crucial step in gene expression analysis via this method. The expression levels of reference genes should be remained constant among cells in different tissues. However, it seems that the location of cells in different tissues might influence their expression. The purpose of this study was to determine whether the source of mesenchymal stem cells (MSCs) has any effect on expression level of three common reference genes (GAPDH, beta-actin and beta 2-microglobulin) in equine marrow- and adipose- derived undifferentiated MSCs and consequently their reliability for comparative qRT-PCR. Materials and methods: Adipose tissue (AT) and bone marrow (BM) samples were harvested from 3 mares. MSCs were isolated and cultured until passage 3 (P3). Total RNA of P3 cells was extracted for cDNA synthesis. The generated cDNAs were analyzed by quantitative real-time PCR. The PCR reactions were ended with a melting curve analysis to verify the specificity of amplicon. Results: The expression levels of GAPDH were significantly different between AT- and BM- derived MSCs (p 0.05). Differences in expression level of beta-actin (P 0.001) and B2M (P 0.006.) between MSCs derived from AT and BM were substantially higher than GAPDH. In addition, the fold change in expression levels of GAPDH, beta-actin and B2M in AT-derived MSCs compared to BM-derived MSCs were 2.38, 6.76 and 7.76, respectively. Conclusion: This study demonstrated that GAPDH and especially beta-actin and B2M express in different levels in equine AT- and BM- derived MSCs. Thus they cannot be considered as reliable reference genes for comparative quantitative gene expression analysis in MSCs derived from equine bone marrow and adipose tissue.