GAPDH, β-actin and β2-microglobulin, As Three Common Reference Genes, Are Not Reliable for Gene Expression Studies in Equine Adipose- and Marrow-derived Mesenchymal Stem Cells

Overview

Journal J Anim Sci Technol

Date 2015 Aug 21

PMID 26290738

Citations 21

Authors

Fatemeh Nazari

Abbas Parham

Adham Fani Maleki

Affiliations

Soon will be listed here.

Abstract

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, β-actin and β2-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 β-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, β-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 β-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.

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References

De Schauwer C, Meyer E, Van de Walle G, Van Soom A . Markers of stemness in equine mesenchymal stem cells: a plea for uniformity. Theriogenology. 2011; 75(8):1431-43. DOI: 10.1016/j.theriogenology.2010.11.008. View

Al-Nbaheen M, Vishnubalaji R, Ali D, Bouslimi A, Al-Jassir F, Megges M . Human stromal (mesenchymal) stem cells from bone marrow, adipose tissue and skin exhibit differences in molecular phenotype and differentiation potential. Stem Cell Rev Rep. 2012; 9(1):32-43. PMC: 3563956. DOI: 10.1007/s12015-012-9365-8. View

Smith R, Korda M, Blunn G, Goodship A . Isolation and implantation of autologous equine mesenchymal stem cells from bone marrow into the superficial digital flexor tendon as a potential novel treatment. Equine Vet J. 2003; 35(1):99-102. DOI: 10.2746/042516403775467388. View

Kern S, Eichler H, Stoeve J, Kluter H, Bieback K . Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells. 2006; 24(5):1294-301. DOI: 10.1634/stemcells.2005-0342. View

Sirover M . New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase. Biochim Biophys Acta. 1999; 1432(2):159-84. DOI: 10.1016/s0167-4838(99)00119-3. View

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3(7):RESEARCH0034. PMC: 126239. DOI: 10.1186/gb-2002-3-7-research0034. View

Nomura T, Huang W, Zhau H, Josson S, Mimata H, Chung L . β2-Microglobulin-mediated signaling as a target for cancer therapy. Anticancer Agents Med Chem. 2013; 14(3):343-52. PMC: 3931390. DOI: 10.2174/18715206113139990092. View

Radtke C, Nino-Fong R, Esparza Gonzalez B, Stryhn H, McDuffee L . Characterization and osteogenic potential of equine muscle tissue- and periosteal tissue-derived mesenchymal stem cells in comparison with bone marrow- and adipose tissue-derived mesenchymal stem cells. Am J Vet Res. 2013; 74(5):790-800. DOI: 10.2460/ajvr.74.5.790. View

Nolan T, Hands R, Bustin S . Quantification of mRNA using real-time RT-PCR. Nat Protoc. 2007; 1(3):1559-82. DOI: 10.1038/nprot.2006.236. View

10.

Russell K, Lacey M, Gilliam J, Tucker H, Phinney D, OConnor K . Clonal analysis of the proliferation potential of human bone marrow mesenchymal stem cells as a function of potency. Biotechnol Bioeng. 2011; 108(11):2716-26. PMC: 3178709. DOI: 10.1002/bit.23193. View

11.

Schmittgen T, Livak K . Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008; 3(6):1101-8. DOI: 10.1038/nprot.2008.73. View

12.

Hjertner B, Olofsson K, Lindberg R, Fuxler L, Fossum C . Expression of reference genes and T helper 17 associated cytokine genes in the equine intestinal tract. Vet J. 2013; 197(3):817-23. DOI: 10.1016/j.tvjl.2013.05.020. View

13.

Amable P, Teixeira M, Carias R, Granjeiro J, Borojevic R . Identification of appropriate reference genes for human mesenchymal cells during expansion and differentiation. PLoS One. 2013; 8(9):e73792. PMC: 3759474. DOI: 10.1371/journal.pone.0073792. View

14.

Ragni E, Vigano M, Rebulla P, Giordano R, Lazzari L . What is beyond a qRT-PCR study on mesenchymal stem cell differentiation properties: how to choose the most reliable housekeeping genes. J Cell Mol Med. 2013; 17(1):168-80. PMC: 3823147. DOI: 10.1111/j.1582-4934.2012.01660.x. View

15.

Burk J, Ribitsch I, Gittel C, Juelke H, Kasper C, Staszyk C . Growth and differentiation characteristics of equine mesenchymal stromal cells derived from different sources. Vet J. 2012; 195(1):98-106. DOI: 10.1016/j.tvjl.2012.06.004. View

16.

Zhu Y, Su Y, Cheng T, Chung L, Shi C . Beta2-microglobulin as a potential factor for the expansion of mesenchymal stem cells. Biotechnol Lett. 2009; 31(9):1361-5. PMC: 2984555. DOI: 10.1007/s10529-009-0027-0. View

17.

Josson S, Nomura T, Lin J, Huang W, Wu D, Zhau H . β2-microglobulin induces epithelial to mesenchymal transition and confers cancer lethality and bone metastasis in human cancer cells. Cancer Res. 2011; 71(7):2600-10. PMC: 3182156. DOI: 10.1158/0008-5472.CAN-10-3382. View

18.

Kim J, Kim S, Han S, Paik S, Hur S, Kim Y . Increased glyceraldehyde-3-phosphate dehydrogenase gene expression in human cervical cancers. Gynecol Oncol. 1998; 71(2):266-9. DOI: 10.1006/gyno.1998.5195. View

19.

Bustin S, Penning L . Improving the analysis of quantitative PCR data in veterinary research. Vet J. 2011; 191(3):279-81. DOI: 10.1016/j.tvjl.2011.06.044. View

20.

Koch T, Berg L, Betts D . Concepts for the clinical use of stem cells in equine medicine. Can Vet J. 2009; 49(10):1009-17. PMC: 2553494. View