Evaluation of reference genes for quantitative real-time PCR in Wharton’s Jelly-derived mesenchymal stem cells after lentiviral transduction and differentiation
Quantitative real time reverse transcription PCR, qRT-PCR, is one of the most important techniques for assessing the level of gene expression. Selecting the correct reference gene to normalize the results is a key step in this method. Inaccurate data can be generated if the correct reference gene is not selected. The level of the expression of reference genes is tissue-variable, and in the case of mesenchymal stem cells (MSC), it can be different depending on the source of their origin. The aim of this study was to select the reference gene for Wharton’s Jelly-derived MSC (WJ- MSC) that were undergoing transduction and differentiation. In this work, the expression of 32 genes was analyzed, of which two (RPS17 and 18S rRNA), which had the most stable expression level, were selected. A comparative analysis of the expression stability of the selected genes was then performed with the genes that are most commonly used in the literature, i.e. β-actin and GAPDH. Next, it was determined that a false picture of the expression level of the studied genes can be obtained when a reference gene with variable expression level is used for normalization. RPS17 and 18S rRNA proved to be the most stable reference genes for the WJ-MSC that had been subjected to the lentiviral transfection procedure followed by differentiation. The expression of β-actin and GAPDH was highly unstable and therefore these genes are not suitable for use as reference genes in studies involving WJ- MSC.
KeywordsMesenchymal stem cells GAPDH Β-actin 18S rRNA RPS17 Reference gene
This work was supported by a Grant from Medical University of Silesia, Katowice, Poland (KNW-1-064/N/9/B).
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Conflict of interest
The authors declare that they have no conflicts of interest.
- 2.Jeon RH, Lee WJ, Son YB, Bharti D, Shivakumar SB, Lee SL, Rho GJ (2019) PPIA, HPRT1, and YWHAZ genes are suitable for normalization of mRNA expression in long-term expanded human mesenchymal stem cells. Biomed Res Int 21(2019):3093545Google Scholar
- 6.Lejkowska R, Kawa MP, Pius-Sadowska E, Rogińska D, Łuczkowska K, Machaliński B, Machalińska A (2019) Preclinical evaluation of long-term neuroprotective effects of BDNF-engineered mesenchymal stromal cells as intravitreal therapy for chronić retinal degeneration in Rd6 mutant mice. Int J Mol Sci 20(3):777CrossRefGoogle Scholar
- 13.Ranera B, Lyahyai J, Romero A, Vazquez FJ, Remacha AR, Bernal ML, Zaragoza P, Rodellar C, Martin-Burriel I (2011) Immunophenotype and gene expression profiles of cell surface markers of mesenchymal stem cells derived from equine bone marrow and adipose tissue. Vet Immunol Immunopathol 144:147–154CrossRefGoogle Scholar
- 14.Nazari F, Parham A, Maleki AF (2005) 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. J Anim Sci 57:18Google Scholar