Kraemer Nadine, Neubert Gerda, Issa Lina, Ninnemann Olaf, Seiler Andrea E M, Kaindl Angela M
Institute of Neuroanatomy and Cell Biology, Charité - Universitätsmedizin Berlin, Germany.
Neurol Res. 2012 Sep;34(7):664-8. doi: 10.1179/1743132812Y.0000000060. Epub 2012 Jun 26.
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.
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.
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.
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.
通过定量实时聚合酶链反应（qPCR）进行基因表达分析是生物学和医学研究中的关键方法。在此，通过对参考基因的平行分析来补偿不同批次和样本之间的差异，这要求参考基因在所有样本和实验过程中具有最稳定的表达，以作为内参标准。实际上，不存在通用的参考基因；相反，假定的参考基因在各种细胞类型中差异很大。这就需要针对每个特定的实验目的评估参考基因，尤其是在发育研究中。本研究的目的是在体内发育的小鼠大脑新皮质以及体外分化的小鼠胚胎干细胞（mESC）中鉴定适合用于基因表达分析的参考基因。
使用C(q)值的四分位数、倍数变化、变异系数（CV）以及最大减去两倍标准差与均值之间的差异作为标准，对五个候选基因Actb、18s、Gapdh、Hprt和RpII在体内和体外的整个发育过程中进行分析，以评估它们的表达稳定性。
我们发现RpII是mESC在整个分化过程中表达最稳定的基因，而在发育中的小鼠新皮质中，Gapdh表现出最高的表达稳定性。
基于我们的结果，我们建议在神经发育背景下进行基因表达分析时，对于mESC使用RpII作为参考基因，对于小鼠新皮质使用Gapdh或Hprt作为参考基因。
