Open Access Highly Accessed Research article

Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells

Liesbeth Vossaert1, Thomas O’Leary2, Christophe Van Neste1, Björn Heindryckx2, Jo Vandesompele3, Petra De Sutter2 and Dieter Deforce1*

Author Affiliations

1 Laboratory for Pharmaceutical Biotechnology, Ghent University, Harelbekestraat 72, Ghent 9000, Belgium

2 Department for Reproductive Medicine, Ghent University Hospital, De Pintelaan 185, Ghent 9000, Belgium

3 Center for Medical Genetics, Ghent University Hospital, De Pintelaan 185, Ghent 9000, Belgium

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BMC Molecular Biology 2013, 14:21  doi:10.1186/1471-2199-14-21

Published: 12 September 2013



Selecting stably expressed reference genes is essential for proper reverse transcription quantitative polymerase chain reaction gene expression analysis. However, this choice is not always straightforward. In the case of differentiating human embryonic stem (hES) cells, differentiation itself introduces changes whereby reference gene stability may be influenced.


In this study, we evaluated the stability of various references during retinoic acid-induced (2 microM) differentiation of hES cells. Out of 12 candidate references, beta-2-microglobulin, ribosomal protein L13A and Alu repeats are found to be the most stable for this experimental set-up.


Our results show that some of the commonly used reference genes are actually not amongst the most stable loci during hES cell differentiation promoted by retinoic acid. Moreover, a novel normalization strategy based on expressed Alu repeats is validated for use in hES cell experiments.

Reverse transcription quantitative PCR; Normalization; Reference genes; Alu repeats; Human embryonic stem cells; Stem cell differentiation