Heinrich Marielle, Lutz-Bonengel Sabine, Matt Katja, Schmidt Ulrike
Institute of Legal Medicine, University of Freiburg, Albertstr 9, 79104 Freiburg, Germany.
Forensic Sci Int Genet. 2007 Jun;1(2):163-9. doi: 10.1016/j.fsigen.2007.01.002. Epub 2007 Mar 23.
Relative quantification of mRNA using quantitative real-time reverse transcription (RT)-PCR is a commonly used method for analysis and comparison of gene expression levels. This method requires a normalisation of data against expression levels of a control gene. In the past, several ubiquitously expressed genes were used as such endogenous controls. When working with human tissue samples obtained during autopsy one has to deal with postmortem intervals of usually more than 10 h. The aim of this study was to investigate whether commonly used endogenous control genes show stability over various postmortem intervals. For this purpose, RNA was extracted from three different human tissues of five postmortem intervals ranging from 15 to 118 h. The Ct values from five commonly used endogenous control genes--beta-actin, B2M, CyPA, TBP, and UBC--were obtained by real-time RT-PCR. Results revealed a relatively high stability of Ct values in skeletal muscle tissue regarding different postmortem intervals. In heart and brain tissues, all endogenous controls were found to be highly variable. B2M appeared to be the least unstable control in this set. Nevertheless, all endogenous controls showed variability in their expression levels regarding both the stability among different tissues and different postmortem intervals. Data obtained in the present study show that postmortem mRNA degradation is a complex process, and that the use of one single endogenous control in gene expression studies of postmortem tissue would lead to erroneous data interpretation. Further studies on this topic should be performed in the future including an increased number of well documented samples.
使用定量实时逆转录（RT）-PCR对mRNA进行相对定量是分析和比较基因表达水平的常用方法。该方法需要根据对照基因的表达水平对数据进行标准化。过去，一些普遍表达的基因被用作这种内参基因。在处理尸检时获得的人体组织样本时，必须应对通常超过10小时的死后间隔时间。本研究的目的是调查常用的内参基因在不同死后间隔时间内是否表现出稳定性。为此，从5个死后间隔时间（15至118小时）的三种不同人体组织中提取RNA。通过实时RT-PCR获得了五个常用内参基因（β-肌动蛋白、B2M、CyPA、TBP和UBC）的Ct值。结果显示，骨骼肌组织中Ct值在不同死后间隔时间内相对稳定。在心脏和脑组织中，所有内参基因都发现有高度的变异性。在这一组中，B2M似乎是最稳定的对照基因。然而，所有内参基因在不同组织间以及不同死后间隔时间的稳定性方面，其表达水平均表现出变异性。本研究获得的数据表明，死后mRNA降解是一个复杂的过程，并且在死后组织的基因表达研究中使用单一内参基因会导致错误的数据解读。未来应就此主题进行进一步研究，包括增加记录完善的样本数量。
