Institute of Legal Medicine, Freiburg University Medical Center, Albertstrasse 9, 79104 Freiburg, Germany.
Int J Legal Med. 2011 Jul;125(4):573-80. doi: 10.1007/s00414-011-0578-1. Epub 2011 May 17.
Messenger RNA (mRNA) profiling in post-mortem human tissue might reveal information about gene expression at the time point of death or close to it. When working with post-mortem human tissue, one is confronted with a natural RNA degradation caused by several parameters which are not yet fully understood. The aims of the present study were to analyse the influence of impaired RNA integrity on the reliability of quantitative gene expression data and to identify ante- and post-mortem parameters that might lead to reduced RNA integrities in post-mortem human brain, cardiac muscle and skeletal muscle tissues. Furthermore, this study determined the impact of several parameters like type of tissue, age at death, gender and body mass index (BMI), as well as duration of agony, cause of death and post-mortem interval on the RNA integrity. The influence of RNA integrity on the reliability of quantitative gene expression data was analysed by generating degradation profiles for three gene transcripts. Based on the deduced cycle of quantification data, this study shows that reverse transcription quantitative polymerase chain reaction (RT-qPCR) performance is affected by impaired RNA integrity. Depending on the transcript and tissue type, a shift in cycle threshold values of up to two cycles was observed. Determining RNA integrity number of 136 post-mortem samples revealed significantly different RNA qualities among the three tissue types with brain revealing significantly lower integrities compared to skeletal and cardiac muscle. The body mass index was found to influence RNA integrity in skeletal muscle tissue (M. iliopsoas). Samples originating from deceased with a BMI > 25 were of significantly lower integrity compared to samples from normal weight donors. Correct data normalisation was found to partly diminish the effects caused by impaired RNA quality. Nevertheless, it can be concluded that in post-mortem tissue with low RNA integrity numbers, the detection of large differences in gene expression activities might still be possible, whereas small expression differences are prone to misinterpretation due to degradation. Thus, when working with post-mortem samples, we recommend generating degradation profiles for all transcripts of interest in order to reveal detection limits of RT-qPCR assays.
信使 RNA（mRNA）在人死后组织中的分析可能会揭示死亡时或接近死亡时的基因表达信息。在使用人死后组织时，由于一些尚未完全理解的参数，会面临自然的 RNA 降解。本研究的目的是分析受损 RNA 完整性对定量基因表达数据可靠性的影响，并确定可能导致人死后大脑、心肌和骨骼肌组织 RNA 完整性降低的生前和死后参数。此外，本研究还确定了一些参数，如组织类型、死亡时的年龄、性别和体重指数（BMI）以及痛苦持续时间、死因和死后间隔对 RNA 完整性的影响。通过为三个基因转录本生成降解曲线来分析 RNA 完整性对定量基因表达数据可靠性的影响。基于推断的定量数据循环，本研究表明逆转录定量聚合酶链反应（RT-qPCR）的性能受到受损 RNA 完整性的影响。根据转录本和组织类型，观察到循环阈值值的变化高达两个循环。对 136 个死后样本的 RNA 完整性数量的测定表明，三种组织类型之间的 RNA 质量存在显著差异，大脑的完整性明显低于骨骼和心肌。发现 BMI 会影响骨骼肌组织（M. iliopsoas）中的 RNA 完整性。来自 BMI > 25 的死者的样本比来自正常体重供体的样本的完整性显著降低。发现正确的数据归一化部分减轻了受损 RNA 质量引起的影响。然而，可以得出结论，在 RNA 完整性数量较低的死后组织中，仍然可能检测到基因表达活性的大差异，而小的表达差异由于降解容易被误解。因此，在使用死后样本时，我们建议为所有感兴趣的转录本生成降解曲线，以揭示 RT-qPCR 检测的检测限。
