Reference genes in the developing murine brain and in differentiating embryonic stem cells.
OBJECTIVE
Gene expression analysis via quantitative real-time PCR (qPCR) is a key approach in biological and medical research. Here, variations between runs and samples are compensated for by in-parallel analysis of reference genes, which require a most stable expression throughout  all samples and experimental procedures to function as internal standards. In reality, there is no universal reference gene; but rather, assumed reference genes vary widely among various cell types. This demands an evaluation of reference genes for each specific experimental purpose, especially  in the case of developmental studies. The aim of the present study was to identify suitable reference genes for gene expression analysis in the developing murine brain neocortex in vivo and in mouse embryonic stem cells (mESC) throughout differentiation in vitro.


METHODS
The five candidate genes Actb, 18s, Gapdh, Hprt, and RpII were analyzed throughout development in vivo and in vitro using the quartiles of C(q) values, fold change, coefficient of variation (CV) and the difference between maximum  minus twofold standard deviation and mean as the criteria to evaluate their expression stability.


RESULTS
We found that RpII was the most stable expressed gene in mESC throughout differentiation, while in the developing murine neocortex Gapdh showed the highest expression  stability.


CONCLUSIONS
Based on our results, we suggest for gene expression analysis in the context of neurodevelopment the usage of RpII as a reference gene for mESC and Gapdh or Hprt for the murine neocortex.
Institute of Neuroanatomy and Cell Biology, Charité - Universitätsmedizin Berlin, Germany. nadine.kraemer@charite.de
Nadine
N
Kraemer
Gerda
G
Neubert
Lina
L
Issa
Olaf
O
Ninnemann
Andrea E M
AE
Seiler
Angela M
AM
Kaindl
D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000166:cytology* / Q000196:embryology / Q000502:physiology; D002454:Cell Differentiation / Q000235:genetics*; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000502:physiology*; D005260:Female; D018507:Gene Expression Regulation, Developmental; D020043:Genes, Essential / Q000235:genetics; D056726:Genetic Association Studies / Q000379:methods; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000096:biosynthesis / Q000235:genetics; D007041:Hypoxanthine Phosphoribosyltransferase / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D011247:Pregnancy; D012319:RNA Polymerase II / Q000096:biosynthesis / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000096:biosynthesis / Q000235:genetics
D000199:Actins; D012337:RNA, Ribosomal, 18S; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D007041:Hypoxanthine Phosphoribosyltransferase; D012319:RNA Polymerase II