Dataset Information

TBP, PPIA, YWHAZ and EF1A1 Are the Most Stably Expressed Genes during Osteogenic Differentiation.

ABSTRACT: RT-qPCR is the gold standard and the most commonly used method for measuring gene expression. Selection of appropriate reference gene(s) for normalization is a crucial part of RT-qPCR experimental design, which allows accurate quantification and reliability of the results. Because there is no universal reference gene and even commonly used housekeeping genes' expression can vary under certain conditions, careful selection of an appropriate internal control must be performed for each cell type or tissue and experimental design. The aim of this study was to identify the most stable reference genes during osteogenic differentiation of the human osteosarcoma cell lines MG-63, HOS, and SaOS-2 using the geNorm, NormFinder, and BestKeeper statistical algorithms. Our results show that TBP, PPIA, YWHAZ, and EF1A1 are the most stably expressed genes, while ACTB, and 18S rRNA expressions are most variable. These data provide a basis for future RT-qPCR normalizations when studying gene expression during osteogenic differentiation, for example, in studies of osteoporosis and other bone diseases.

SUBMITTER: Franko N

PROVIDER: S-EPMC9025278 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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TBP, PPIA, YWHAZ and EF1A1 Are the Most Stably Expressed Genes during Osteogenic Differentiation.

Franko Nina N Vrščaj Lucija Ana LA Zore Taja T Ostanek Barbara B Marc Janja J Lojk Jasna J

International journal of molecular sciences 20220412 8

RT-qPCR is the gold standard and the most commonly used method for measuring gene expression. Selection of appropriate reference gene(s) for normalization is a crucial part of RT-qPCR experimental design, which allows accurate quantification and reliability of the results. Because there is no universal reference gene and even commonly used housekeeping genes' expression can vary under certain conditions, careful selection of an appropriate internal control must be performed for each cell type or ...[more]

PMID: 35457075

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Project description:BackgroundMesenchymal stem cells derived from human tissues have been widely used for tissue regeneration because of their strong self-renewal capacity and multi-potential properties. Autophagy plays a vital role in maintaining bone homeostasis. However, the mechanism underlying this role for autophagy in the osteogenic differentiation of mesenchymal stem cells remains to be elucidated.MethodsTwo microarray datasets were downloaded from the GEO database. Fourteen bone marrow mesenchymal stem cell samples comprising control and induction groups were selected to identify differentially expressed autophagy-related genes via multiple bioinformatics approaches, followed by functional analysis. Interactions among differentially expressed autophagy genes, miRNAs, and transcription factors were analyzed and visualized using Cytoscape software. The association between hub differentially expressed genes and autophagy was validated by qRT-PCR.ResultsTen autophagy-related genes (including VPS8, NDRG4, and CYBB) were identified as osteogenic hub genes. Correlation analysis revealed that CYBB was highly correlated with the sensitivity to multiple drugs, such as imexon, megestrol acetate, and isotretinoin. The regulatory network displayed a complex connection among miRNAs, transcription factors, and differentially expressed autophagy genes. Friends' analysis showed that NDRG4 was highly closely related to other hub genes (P < 0.05). Furthermore, NDRG4 expression was downregulated in the induction group (P < 0.01). NDRG4 was significantly correlated with infiltrating immune cells, including monocytes, eosinophils, type 17 T helper cells, neutrophils, activated CD8 T cells, and immature B cells. Levels of the 10 autophagy-related genes (including VPS8, NDRG4, and CYBB) were successfully validated based on in vitro experiments.ConclusionWe identified candidate molecules to further investigate their functions in osteogenesis, providing novel insights into the role of autophagy in mesenchymal stem cell differentiation.

Dataset Information

TBP, PPIA, YWHAZ and EF1A1 Are the Most Stably Expressed Genes during Osteogenic Differentiation.

Publications

<i>TBP</i>, <i>PPIA</i>, <i>YWHAZ</i> and <i>EF1A1</i> Are the Most Stably Expressed Genes during Osteogenic Differentiation.

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