Int. J. Dev. Biol. 50: 627 - 635 (2006)

https://doi.org/10.1387/ijdb.052130ew

Vol 50, Issue 7

Selection of reference genes in mouse embryos and in differentiating human and mouse ES cells

Published: 1 August 2006

Erik Willems¹, Ileana Mateizel², Caroline Kemp¹, Greet Cauffman², Karen Sermon² and Luc Leyns*^,1

¹Lab for Cell Genetics, Vrije Universiteit Brussel (VUB) and SUP>2Research Center for Reproduction and Genetics, University Hospital and Medical School of the Vrije Universiteit Brussel (AZ-VUB), Brussels, Belgium

Abstract

Embryonic Stem (ES) cells have the potential to form every cell of the body and thus are of great promise for tissue transplantation. One of the rising techniques that allows studying the differentiation state of ES cells is quantitative RT-PCR (qRT-PCR). When relative quantification by qRT-PCR is applied, accurate normalization is necessary, since differentiated embryonic stem cells and developing embryos contain heterogeneous cell populations. Corrections for variations in the qRT-PCR reaction are needed to allow comparisons between different samples. We applied the normalization tools geNorm and Normfinder to ten reference genes identifying the most stable ones for relative quantification of gene expression during differentiation of human ES cells, as well as in differentiated mouse ES cells and in the developing mouse embryo. For relative quantification by qRT-PCR in these systems, we advise to use normalization factors based on multiple stable reference genes. However, when the use of several reference genes would be unpractical, a single reference gene in each experimental setup could be sufficient. When looking for single stable reference genes, beta-actin works best in both mouse embryo and ES cell experiments and glyceraldehyde-3-phosphate-dehydrogenase can be applied in both mouse and human ES cell experiments.

Keywords

embryonic stem cell, embryo, quantitative RT-PCR, reference gene