Frericks Markus, Esser Charlotte
Institut für Umweltmedizinische Forschung (IUF) at the Heinrich Heine-University of Düsseldorf, Division of Molecular Immunology, Auf'm Hennekamp 50, 40225 Düsseldorf, Germany.
Biochim Biophys Acta. 2008 Dec;1779(12):830-7. doi: 10.1016/j.bbagrm.2008.08.007. Epub 2008 Aug 27.
Quantitative and semi-quantitative analysis of gene transcripts requires normalization to RNA-input and/or invariantly expressed house-keeping genes. Currently, only a limited choice of reference genes exists, such as GAPDH(1), beta-actin, or HPRT, whose transcription levels may be less stable than previously thought. We used the meta-database NC-GED, which we had derived from 1968 published murine expression profiles to identify genes with (i) low inter-tissue expression variability and (ii) great stability over 312 conditions, such as experimental drug treatment, age or differentiation. We identified 276 novel genes with "house-keeping" characteristics, including many genes for ribosomal proteins, and aryl-hydrocarbon receptor-interacting protein. Most genes yielded medium to strong fluorescence intensity on the arrays, a relative measure for their cellular expression. We validated the invariant expression levels of eight of the house-keeper candidates in lymph nodes, thymus, liver, kidney and brain of four different mouse strains. In addition, comparative analysis showed the superiority of multiple over single standardization. Caution against established reference genes is justified. The new panel of reference genes is useful for a flexible selection of reference genes in gene expression studies.
基因转录本的定量和半定量分析需要对RNA输入量和/或恒定表达的管家基因进行标准化。目前，可供选择的参考基因有限，如甘油醛-3-磷酸脱氢酶（GAPDH）(1)、β-肌动蛋白或次黄嘌呤磷酸核糖转移酶（HPRT），其转录水平可能不如先前认为的那样稳定。我们使用了元数据库NC-GED，该数据库源自1968年发表的小鼠表达谱，以鉴定具有以下特征的基因：（i）组织间表达变异性低；（ii）在312种条件下（如实验性药物治疗、年龄或分化）具有高度稳定性。我们鉴定出276个具有“管家”特征的新基因，包括许多核糖体蛋白基因和芳烃受体相互作用蛋白。大多数基因在芯片上产生中等至强荧光强度，这是其细胞表达的相对指标。我们在四种不同小鼠品系的淋巴结、胸腺、肝脏、肾脏和大脑中验证了八个管家基因候选基因的恒定表达水平。此外，比较分析表明多重标准化优于单一标准化。对既定参考基因保持谨慎是合理的。新的参考基因面板有助于在基因表达研究中灵活选择参考基因。
