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Journal of Ovarian Research

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Open Access 01-12-2013 | Research

Normalizing to GADPH jeopardises correct quantification of gene expression in ovarian tumours – IPO8 and RPL4 are reliable reference genes

Authors: Zuzana Kolkova, Arsen Arakelyan, Bertil Casslén, Stefan Hansson, Eva Kriegova

Published in: Journal of Ovarian Research | Issue 1/2013

Abstract

Background

To ensure a correct interpretation of results obtained with quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR), it is critical to normalize to a reference gene with stable mRNA expression in the tissue of interest. GADPH is widely used as a reference gene in ovarian tumour studies, although lacking tissue-specific stability. The aim of this study was to identify alternative suitable reference genes for RT-qPCR studies on benign, borderline, and malignant ovarian tumours.

Methods

We assayed mRNA levels for 13 potential reference genes – ABL1, ACTB, CDKN1A, GADPH, GUSB, HPRT1, HSP90AB, IPO8, PPIA, RPL30, RPL4, RPLPO, and TBP –with RT-qPCR in 42 primary ovarian tumours, using commercially pre-designed RT-qPCR probes. Expression stability was subsequently analysed with four different statistical programs (GeNorm, NormFinder, BestKeeper, and the Equivalence test).

Results

Expression of IPO8, RPL4, TBP, RPLPO, and ACTB had the least variation in expression across the tumour samples according to GeNorm, NormFinder, and BestKeeper. The Equivalence test found variation in expression within a 3-fold expression change between tumour groups for: IPO8, RPL40, RPL30, GUSB, TBP, RPLPO, ACTB, ABL1, and CDKN1A. However, only IPO8 satisfied at a 2-fold change as a cut-off. Overall, IPO8 and RPL4 had the highest, whereas GADPH and HPRT1 the lowest expression stability. Employment of suitable reference genes (IPO8, RPL4) in comparison with unsuitable ones (GADPH, HPRT1), demonstrated divergent influence on the mRNA expression pattern of our target genes − GPER and uPAR.

Conclusions

We found IPO8 and RPL4 to be suitable reference genes for normalization of target gene expression in benign, borderline, and malignant ovarian tumours. Moreover, IPO8 can be recommended as a single reference gene. Neither GADPH nor HPRT1 should be used as reference genes in studies on ovarian tumour tissue.

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Title: Normalizing to GADPH jeopardises correct quantification of gene expression in ovarian tumours – IPO8 and RPL4 are reliable reference genes
Authors: Zuzana Kolkova
Arsen Arakelyan
Bertil Casslén
Stefan Hansson
Eva Kriegova
Publication date: 01-12-2013
Publisher: BioMed Central
Published in: Journal of Ovarian Research / Issue 1/2013
Electronic ISSN: 1757-2215
DOI: https://doi.org/10.1186/1757-2215-6-60

At a glance: The STEP trials

Springer Medicine

Normalizing to GADPH jeopardises correct quantification of gene expression in ovarian tumours – IPO8 and RPL4 are reliable reference genes

Abstract

Background

Methods

Results

Conclusions

At a glance: The STEP trials

Springer Medicine

Abstract

Background

Methods

Results

Conclusions

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