Real-time PCR detection of five different "endogenous control gene" transcripts in forensic autopsy material
Real-time PCR detection of five different "endogenous control gene" transcripts in forensic autopsy material
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DOI：
10.1016/j.fsigen.2007.01.002
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发表时间：
2007-06-01
影响因子：
3.1
通讯作者：
Schmidt, Ulrike
中科院分区：
文献类型：
--
作者：
Heinrich, Marielle;Lutz-Bonengel, Sabine;Schmidt, Ulrike
实时定量逆转录聚合酶链式反应是基因表达水平分析和比较的常用方法。这种方法需要对照对照基因的表达水平对数据进行标准化。在过去，几个普遍表达的基因被用作这样的内源对照。当处理在尸检中获得的人类组织样本时，人们必须处理通常超过10小时的死后间隔。本研究的目的是调查常用的内源控制基因在不同的死后间隔内是否表现出稳定性。为此，从死后15~118h的5个不同的人体组织中提取RNA，通过实时荧光RT-PCR获得了5个常用的内源调控基因--β-肌动蛋白、B2M、CyPA、TBP和UBC的C-t值。结果表明，不同的死后时间骨骼肌组织中的C1值具有较高的稳定性。在心脏和脑组织中，所有内源性对照都被发现是高度可变的。B2M似乎是这组中不稳定程度最低的。然而，所有内源对照的表达水平在不同组织之间和不同死后时间的稳定性方面都表现出差异。本研究获得的数据表明，死后mRNA的降解是一个复杂的过程，在死后组织的基因表达研究中使用单一的内源对照会导致错误的数据解释。今后应对这一专题进行进一步研究，包括增加有充分记录的样本数量。(C)2007爱思唯尔爱尔兰有限公司。保留所有权利。
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 C-t values from five commonly used endogenous control genes -beta-actin, B2M, CyPA, TBP, and UBC were obtained by real-time RT-PCR.Results revealed it relatively high stability of C, 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. (c) 2007 Elsevier Ireland Ltd. All rights reserved.