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Open Chemistry

formerly Central European Journal of Chemistry


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Volume 11, Issue 3

Issues

Volume 13 (2015)

Selection of reference genes for quantitative real-time PCR evaluation of chronic erythropoietin treatment effect on the SH-SY5Y and PC12 cells

Klemen Španinger
  • Center for Functional Genomics and Bio-chips, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, 1000, Ljubljana, Slovenia
  • Slovenia and DiaGenomi Ltd, 1000, Ljubljana, Slovenia
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Arthur Sytkowski
  • Division of Hematology and Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, FA-824, Boston, MA, 02215, USA
  • Oncology Therapeutic Area, Therpeutic Delivery Unit, Quintiles Transnational, Arlington, MA, 02476, USA
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Nataša Debeljak
  • Center for Functional Genomics and Bio-chips, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, 1000, Ljubljana, Slovenia
  • Slovenia and DiaGenomi Ltd, 1000, Ljubljana, Slovenia
  • Medical Centre for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, 1000, Ljubljana, Slovenia
  • Email
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
Published Online: 2012-12-28 | DOI: https://doi.org/10.2478/s11532-012-0164-5

Abstract

Abstract The quantitative real-time polymerase chain reaction (qPCR) is a sensitive technique for examining the influence of erythropoietin (Epo) on gene expression. A critical and fundamental step for data analysis is the selection of and normalization to the optimal reference gene(s). We identified appropriate reference gene(s) among 32 genes during chronic recombinant human Epo (rHuEpo) treatment of SH-SY5Y cells using TaqMan human Express Endogenous Control Plate. Expression stability of the selected reference gene (RPLP) was retested with qPCR, together with two commonly used reference genes (GAPDH, ACTB) and six genes of interest (EPOR, EPO, STAT5B, STAT5A, JUN, AKT). In PC12 cells, three commonly used reference genes (Gapdh, CycA and Ywhaz) and seven genes of interest (EpoR, Epo, Stat5b, Stat5a, Jun, Akt, Fos) were evaluated. For the evaluation of expression stability, geNorm, NormFinder and BestKeeper software were used. All three gave similar results. We demonstrated that among the housekeeping genes, RPLP in SH-SY5Y and CycA and Ywhaz in PC12 are the most stable genes. Additionally, we showed that normalization with GAPDH gave misleading results compared to normalization with geNorm. In conclusion, selection of the appropriate normalization gene(s) is crucial for correct interpretation of rHuEpo treatment results. Graphical abstract

Keywords: Erythropoietin (Epo); Chronic treatment; Quantitative real-time PCR; Reference gene selection; Normalization

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About the article

Published Online: 2012-12-28

Published in Print: 2013-03-01


Citation Information: Open Chemistry, Volume 11, Issue 3, Pages 348–357, ISSN (Online) 2391-5420, DOI: https://doi.org/10.2478/s11532-012-0164-5.

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© 2012 Versita Warsaw. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

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