Kosheverova Vera, Schwarz Alexander, Kamentseva Rimma, Kharchenko Marianna, Kornilova Elena
Laboratory of Intracellular Membranes Dynamics, Institute of Cytology of the Russian Academy of Sciences, 194064 Saint Petersburg, Russia.
Laboratory of Molecular Mechanisms of Neural Interactions, Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences, 194223 Saint Petersburg, Russia.
Front Biosci (Schol Ed). 2024 Dec 25;16(4):26. doi: 10.31083/j.fbs1604026.
Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful tool for analysing target gene expression in biological samples. To achieve reliable results by RT-qPCR, the most stable reference genes must be selected for proper data normalisation, particularly when comparing cells of different types. We aimed to choose the least variable candidate reference genes among eight housekeeping genes tested within a set of human cancer cell lines (HeLa, MCF-7, SK-UT-1B, A549, A431, SK-BR-3), as well as four lines of normal, non-malignant mesenchymal stromal cells (MSCs) of different origins.
The reference gene stability was evaluated using four algorithms (BestKeeper, NormFinder, geNorm and the comparative ΔCt method) and ranked with the RefFinder web-based tool.
We found increased variability in the housekeeping genes' expression in the cancer cell lines compared to that in normal MSCs. and were identified as the most suitable reference genes in cancer cells, while and were the most suitable in MSCs. and were shown to be the least variable genes when analysing normal and cancer cell lines together. Epidermal growth factor receptor (EGFR) mRNA relative expression was normalised by the three most stable or three least stable reference genes to demonstrate the reliability of reference genes validation.
We analysed and selected stable reference genes for RT-qPCR analysis in the wide panel of cancer cell lines and MSCs. The study provides a reliable tool for future research concerning the expression of genes involved in various intracellular signalling pathways and emphasises the need for careful selection of suitable references before analysing target gene expression.
实时逆转录定量聚合酶链反应（RT-qPCR）是分析生物样品中靶基因表达的有力工具。为了通过RT-qPCR获得可靠结果，必须选择最稳定的内参基因进行适当的数据归一化，尤其是在比较不同类型细胞时。我们旨在从一组人类癌细胞系（HeLa、MCF-7、SK-UT-1B、A549、A431、SK-BR-3）以及四种不同来源的正常非恶性间充质基质细胞（MSC）系中测试的八个管家基因中选择变异性最小的候选内参基因。
使用四种算法（BestKeeper、NormFinder、geNorm和比较ΔCt法）评估内参基因的稳定性，并通过基于网络的RefFinder工具进行排名。
我们发现与正常MSC相比，癌细胞系中管家基因表达的变异性增加。在癌细胞中，[具体基因1]和[具体基因2]被确定为最合适的内参基因，而在MSC中，[具体基因3]和[具体基因4]是最合适的。在同时分析正常细胞系和癌细胞系时，[具体基因5]和[具体基因6]显示为变异性最小的基因。通过三个最稳定或三个最不稳定的内参基因对表皮生长因子受体（EGFR）mRNA相对表达进行归一化，以证明内参基因验证的可靠性。
我们分析并选择了用于RT-qPCR分析的稳定内参基因，用于广泛的癌细胞系和MSC。该研究为未来关于参与各种细胞内信号通路的基因表达的研究提供了可靠工具，并强调在分析靶基因表达之前仔细选择合适内参的必要性。
