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

• Authors: Klemen Španinger , Arthur Sytkowski , Nataša Debeljak
• Languages of publication: EN

Abstracts

EN

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

EN

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

• Publisher: De Gruyter Open
• Journal: Open Chemistry
• Year: 2013
• Volume: 11
• Issue: 3
• Pages: 348-357

Dates

published

1 - 3 - 2013

online

28 - 12 - 2012

Contributors

author

Klemen Španinger

author

Arthur Sytkowski

author

Nataša Debeljak

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Identifiers

DOI

10.2478/s11532-012-0164-5