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UBC and YWHAZ as suitable reference genes for accurate normalisation of gene expression using MCF7, HCT116 and HepG2 cell lines

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Abstract

Relative quantification of in vitro gene expression using real-time PCR requires stably expressed reference gene for normalisation. In this study, total RNA from MCF7, HCT116 and HepG2 cells were extracted and converted to cDNA using commercially available kit, and real-time PCR was then performed to analyse the expression levels of twelve reference genes to select the most ideal reference gene for accurate normalisation in gene expression study. geNorm and NormFinder software were used to analyse the stabilities of the reference genes, which showed a wide range of Ct values. The geNorm analysis showed the following ranking for stability of genes: UBC, YWHAZ > RPLP > TBP > ACTB > HPRT1 > PPIA > GAPDH > GUSB > B2M > TUBB > RRN18S. A similar ranking of reference genes was obtained by NormFinder, and the four most stable reference genes were identical using both approaches. UBC and YWHAZ were proposed to be the two most suitable reference genes based on the above analyses. To further assess the stabilities of the UBC and YWHAZ in a formal experiment, MCF7, HCT116 and HepG2 cell lines were subjected to treatments with 5-aza-dC and TSA. Both UBC and YWHAZ exhibited stable expression levels across control and treatment groups. Therefore, we propose that UBC and YWHAZ are the two most suitable reference genes for our gene expression studies using MCF7, HCT116 and HepG2 cell lines.

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Acknowledgments

This work was funded by a Fundamental Research Grant (203/PPSK/6171103) from the Ministry of Higher Education, and Chua Siang Ling was supported by a fellowship from the Institute of Postgraduate Studies, Universiti Sains Malaysia. We also thank the technical support from UPMS, Culture and Biomedicine Laboratories, School of Health Sciences, Universiti Sains Malaysia.

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Authors and Affiliations

  1. School of Health Sciences, Universiti Sains Malaysia, 16150, Kubang Kerian, Kelantan, Malaysia

    Siang Ling Chua, Wei Cun See Too & Ling Ling Few

  2. Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800, USM, Penang, Malaysia

    Boon Yin Khoo

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  1. Siang Ling Chua
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  2. Wei Cun See Too
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  3. Boon Yin Khoo
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Correspondence to Boon Yin Khoo or Ling Ling Few.

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Chua, S.L., See Too, W.C., Khoo, B.Y. et al. UBC and YWHAZ as suitable reference genes for accurate normalisation of gene expression using MCF7, HCT116 and HepG2 cell lines. Cytotechnology 63, 645–654 (2011). https://doi.org/10.1007/s10616-011-9383-4

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  • DOI: https://doi.org/10.1007/s10616-011-9383-4

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