Passmore Margaret, Nataatmadja Maria, Fraser John F
Department of Medicine, The Prince Charles Hospital, Chermside, Queensland, Australia.
BMC Mol Biol. 2009 Jul 23;10:72. doi: 10.1186/1471-2199-10-72.
Heart and lung transplantation is frequently the only therapeutic option for patients with end stage cardio respiratory disease. Organ donation post brain stem death (BSD) is a pre-requisite, yet BSD itself causes such severe damage that many organs offered for donation are unusable, with lung being the organ most affected by BSD. In Australia and New Zealand, less than 50% of lungs offered for donation post BSD are suitable for transplantation, as compared with over 90% of kidneys, resulting in patients dying for lack of suitable lungs. Our group has developed a novel 24 h sheep BSD model to mimic the physiological milieu of the typical human organ donor. Characterisation of the gene expression changes associated with BSD is critical and will assist in determining the aetiology of lung damage post BSD. Real-time PCR is a highly sensitive method involving multiple steps from extraction to processing RNA so the choice of housekeeping genes is important in obtaining reliable results. Little information however, is available on the expression stability of reference genes in the sheep pulmonary artery and lung. We aimed to establish a set of stably expressed reference genes for use as a standard for analysis of gene expression changes in BSD.
We evaluated the expression stability of 6 candidate normalisation genes (ACTB, GAPDH, HGPRT, PGK1, PPIA and RPLP0) using real time quantitative PCR. There was a wide range of Ct-values within each tissue for pulmonary artery (15-24) and lung (16-25) but the expression pattern for each gene was similar across the two tissues. After geNorm analysis, ACTB and PPIA were shown to be the most stably expressed in the pulmonary artery and ACTB and PGK1 in the lung tissue of BSD sheep.
Accurate normalisation is critical in obtaining reliable and reproducible results in gene expression studies. This study demonstrates tissue associated variability in the selection of these normalisation genes in BSD sheep and underlines the importance of selecting the correct reference genes for both the animal model and tissue studied.
心肺移植常常是终末期心肺疾病患者的唯一治疗选择。脑死亡后器官捐献是一个先决条件，但脑死亡本身会造成严重损害，以至于许多用于捐献的器官无法使用，肺是受脑死亡影响最严重的器官。在澳大利亚和新西兰，脑死亡后提供的肺中，只有不到50%适合移植，而肾脏的这一比例超过90%，这导致患者因缺乏合适的肺而死亡。我们的研究小组开发了一种新型的24小时绵羊脑死亡模型，以模拟典型人类器官捐献者的生理环境。表征与脑死亡相关的基因表达变化至关重要，这将有助于确定脑死亡后肺损伤的病因。实时聚合酶链反应是一种高度灵敏的方法，涉及从RNA提取到处理的多个步骤，因此管家基因的选择对于获得可靠结果很重要。然而，关于绵羊肺动脉和肺中参考基因的表达稳定性，几乎没有相关信息。我们旨在建立一组稳定表达的参考基因，作为分析脑死亡中基因表达变化的标准。
我们使用实时定量聚合酶链反应评估了6个候选标准化基因（ACTB、GAPDH、HGPRT、PGK1、PPIA和RPLP0）的表达稳定性。在肺动脉（15 - 24）和肺（16 - 25）的每个组织中，Ct值范围很广，但每个基因在两个组织中的表达模式相似。经过geNorm分析，ACTB和PPIA在肺动脉中表达最稳定，ACTB和PGK1在脑死亡绵羊的肺组织中表达最稳定。
在基因表达研究中，准确的标准化对于获得可靠且可重复的结果至关重要。本研究表明，在脑死亡绵羊中选择这些标准化基因存在组织相关的变异性，并强调了为所研究的动物模型和组织选择正确参考基因的重要性。
