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.

中文翻译：

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慢病毒转导和分化后沃顿氏胶冻源间充质干细胞中实时荧光定量PCR参考基因的评估。

实时定量逆转录PCR（qRT-PCR）是评估基因表达水平的最重要技术之一。选择正确的参考基因以标准化结果是该方法的关键步骤。如果未选择正确的参考基因，可能会产生不正确的数据。参考基因的表达水平是组织可变的，在间充质干细胞（MSC）的情况下，其来源可能不同。这项研究的目的是为沃顿商学院的果冻来源的MSC（WJ-MSC）进行转导和分化选择参考基因。在这项工作中，分析了32个基因的表达，其中选择了两个具有最稳定表达水平的基因（RPS17和18S rRNA）。然后，与文献中最常用的基因（即β-肌动蛋白和GAPDH）进行所选基因表达稳定性的比较分析。接下来，当使用具有可变表达水平的参考基因进行归一化时，确定可以获得所研究基因的表达水平的假图片。事实证明，RPS17和18S rRNA是WJ-MSC最稳定的参考基因，已经经历了慢病毒转染程序并进行了分化。β-肌动蛋白和GAPDH的表达高度不稳定，因此这些基因不适合用作涉及WJ-MSC的研究的参考基因。当使用具有可变表达水平的参考基因进行归一化时，可以确定获得的研究基因表达水平的假象。RPS17和18S rRNA被证明是WJ-MSC的最稳定的参考基因，已经经历了慢病毒转染程序并进行了分化。β-肌动蛋白和GAPDH的表达高度不稳定，因此这些基因不适合用作涉及WJ-MSC的研究的参考基因。当使用具有可变表达水平的参考基因进行归一化时，可以确定获得的研究基因表达水平的假象。RPS17和18S rRNA被证明是WJ-MSC的最稳定的参考基因，已经经历了慢病毒转染程序并进行了分化。β-肌动蛋白和GAPDH的表达高度不稳定，因此这些基因不适合用作涉及WJ-MSC的研究的参考基因。