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Identification of appropriate reference genes for human mesenchymal cells during expansion and differentiation.

ABSTRACT: BACKGROUND: Quantitative real time polymerase chain reaction (qPCR) is an extremely powerful technique for monitoring gene expression. The quantity of the messenger ribonucleic acids (mRNA) of interest should be normalized using a reference gene, in order to avoid unreliable results originated by the obtained RNA quality and quantity, manipulation errors and inhibitory contaminants. A reference gene is any gene that is stably and consistently expressed under the conditions being studied. Completely false data can be generated if a reference gene is not chosen adequately. RESULTS: In the present study, we compared expression levels of five putative reference genes (HPRT1, ACTB, GAPDH, RPL13A and B2M) in primary cultures of four different human cells: mesenchymal stromal cells obtained from bone marrow, adipose tissue or umbilical cord Wharto?s Jelly, and dermal fibroblasts, under different expansion and differentiation conditions. We observed that reference genes are not the same for different cells under the same culture conditions. CONCLUSION: Most stable reference genes under our experimental conditions were: RPL13A for adipose tissue- and Wharto?s Jelly-derived mesenchymal stromal cells, and HPRT1 for bone marrow-derived mesenchymal stromal cells and dermal fibroblasts. ACTB was the most unstable gene when evaluating adipose tissue- and Wharto?s Jelly-derived mesenchymal stromal cells, whilst GAPDH and B2M were the most unstable genes for bone marrow-derived mesenchymal stromal cells and dermal fibroblasts, respectively.

SUBMITTER: Amable PR

PROVIDER: S-EPMC3759474 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Identification of appropriate reference genes for human mesenchymal cells during expansion and differentiation.

Amable Paola Romina PR Teixeira Marcus Vinicius Telles MV Carias Rosana Bizon Vieira RB Granjeiro José Mauro JM Borojevic Radovan R

PloS one 20130902 9

<h4>Background</h4>Quantitative real time polymerase chain reaction (qPCR) is an extremely powerful technique for monitoring gene expression. The quantity of the messenger ribonucleic acids (mRNA) of interest should be normalized using a reference gene, in order to avoid unreliable results originated by the obtained RNA quality and quantity, manipulation errors and inhibitory contaminants. A reference gene is any gene that is stably and consistently expressed under the conditions being studied. ...[more]

PMID: 24023904

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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

Identification of appropriate reference genes for human mesenchymal cells during expansion and differentiation.

Publications

Identification of appropriate reference genes for human mesenchymal cells during expansion and differentiation.

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