Identification of valid endogenous control genes for determining gene expression in C6 glioma cell line treated with conditioned medium from adipose-derived stem cell

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• 作者：I.C. Iser ; R.P. de Campos ; A.P.S. Bertoni ; M.R. Wink ; ^{marciawink@yahoo.com.br" class="auth_mail" title="E-mail the corresponding author} ; ^{mwink@ufcspa.edu.br" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Real time RT-qPCR ; Adipose-derived stem cell ; ADSCs ; C6 glioma cell line ; Gene expression ; Conditioned medium
• 刊名：Biomedicine & Pharmacotherapy
• 出版年：2015
• 出版时间：October 2015
• 年：2015
• 卷：75
• 期：Complete
• 页码：75-82
• 全文大小：1884 K

文摘

There is growing evidence that mesenchymal stem cells (MSCs) can be important players in the tumor microenvironment. They can affect the glioma progression through the modulation of different genes. This modulation can be evaluated through a very useful model, treating the tumor cells with MSC-conditioned medium. However, for an accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is a prerequisite.

Methods

We performed a systematic review in an attempt to find a reference gene to use when analyzing gene expression in C6 glioma cells lines. Considering that we were not able to find a reference gene originated by an appropriate validation, in this study we evaluated candidate genes to be used as reference gene in C6 cells under different treatments with adipose-derived stem cells conditioned medium (CM-ADSCs). 尾-actin (ACTB); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine-guanine phosphoribosyltransferase I (HPRT-1); TATA box binding protein (TBP) and beta-2-microglobulin (B2 M) were evaluated by real-time reverse transcription PCR (RT-qPCR). The mean Cq, the maximum fold change (MFC) and NormFinder software were used for reference gene evaluation and selection.

Results

The GAPDH and ACTB genes have been the most widely used reference genes to normalize among the different investigated genes in our review, however, controversially these genes underwent a substantial variability among the genes evaluated in the present work. Individually, TBP gene was more stable when compared with other genes analyzed and the combination of TBP and HPRT-1 was even more stable.

Conclusion

These results evidence the importance of appropriate validation of reference genes before performing qPCR experiments. Besides, our data will contribute with researchers that work analyzing the role of ADSCs in glioma microenvironment through gene expression.