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

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.