Fonseca Costa Sara S, Wegmann Daniel, Ripperger Jürgen A
Department of Biology, University of Fribourg, Fribourg, Switzerland.
Swiss Institute of Bioinformatics, University of Lausanne, Lausanne, Switzerland.
PLoS One. 2017 Jan 9;12(1):e0169615. doi: 10.1371/journal.pone.0169615. eCollection 2017.
The expression of some genes is affected by age. To detect such age-related changes, their expression levels are related to constant marker genes. However, transcriptional noise increasing with advancing age renders difficult the identification of real age-related changes because it may affect the marker genes as well. Here, we report a selection procedure for genes appropriate to normalise the mouse liver transcriptome under various conditions including age. These genes were chosen from an initial set of 16 candidate genes defined based on a RNA-sequencing experiment and published literature. A subset of genes was selected based on rigorous statistical assessment of their variability using both RNA-sequencing and Nanostring hybridization experiments. The robustness of these marker genes was then verified by the analysis of 130 publicly available data sets using the mouse liver transcriptome. Altogether, a set of three genes, Atp5h, Gsk3β, and Sirt2 fulfilled our strict selection criteria in all assessments, while four more genes, Nono, Tprkb, Tspo, and Ttr passed all but one assessment and were included into the final set of marker genes to enhance robustness of normalisation against outliers. Using the geometric mean of expression of the genes to normalise Nanostring hybridization experiments we reliably identified age-related increases in the expression of Casein kinase 1δ and 1ϵ, and Sfpq, while the expression of the glucose transporter Glut2 decreased. The age-related changes were verified by real-time PCR and Western blot analysis. As conclusion, proper normalisation enhances the robustness of quantitative methods addressing age-related changes of a transcriptome.
一些基因的表达受年龄影响。为了检测这种与年龄相关的变化，它们的表达水平与恒定的标记基因相关。然而，随着年龄增长转录噪声增加，使得难以识别真正的与年龄相关的变化，因为它可能也会影响标记基因。在此，我们报告了一种在包括年龄在内的各种条件下对小鼠肝脏转录组进行标准化的合适基因的筛选程序。这些基因是从基于RNA测序实验和已发表文献定义的16个候选基因的初始集合中选择的。基于对使用RNA测序和纳米串杂交实验的变异性的严格统计评估，选择了一个基因子集。然后通过使用小鼠肝脏转录组分析130个公开可用数据集来验证这些标记基因的稳健性。总之，一组三个基因，Atp5h、Gsk3β和Sirt2在所有评估中都符合我们严格的选择标准，而另外四个基因，Nono、Tprkb、Tspo和Ttr除一项评估外通过了所有评估，并被纳入最终的标记基因集，以增强针对异常值的标准化稳健性。使用这些基因表达的几何平均值对纳米串杂交实验进行标准化，我们可靠地鉴定出酪蛋白激酶1δ和1ε以及Sfpq表达的与年龄相关的增加，而葡萄糖转运蛋白Glut2的表达下降。通过实时PCR和蛋白质印迹分析验证了与年龄相关的变化。总之，适当的标准化增强了定量方法针对转录组与年龄相关变化的稳健性。
