RNA integrity in post-mortem samples: influencing parameters and implications on RT-qPCR assays
RNA integrity in post-mortem samples: influencing parameters and implications on RT-qPCR assays
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DOI：
10.1007/s00414-011-0578-1
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发表时间：
2011-07-01
影响因子：
2.1
通讯作者：
Vennemann, Marielle
中科院分区：
文献类型：
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作者：
Koppelkamm, Antje;Vennemann, Benedikt;Vennemann, Marielle
人类死后组织中的信使RNA（mRNA）谱可能揭示死亡或接近死亡时基因表达的信息。当处理死后组织时，人们面临着由几个参数引起的自然RNA降解，这些参数尚未完全了解。本研究的目的是分析定量基因表达数据的可靠性受损的RNA完整性的影响，并确定前和死后的参数，可能会导致减少死后人脑，心肌和骨骼肌组织的RNA完整性。此外，这项研究确定了几个参数的影响，如组织类型，死亡年龄，性别和体重指数（BMI），以及痛苦的持续时间，死亡原因和死后间隔对RNA完整性的影响。通过生成三种基因转录物的降解谱，分析了RNA完整性对定量基因表达数据可靠性的影响。基于定量数据的推导循环，本研究表明，逆转录定量聚合酶链反应（RT-qPCR）的性能受到RNA完整性受损的影响。根据转录本和组织类型，观察到循环阈值的偏移高达两个循环。确定136个死后样本的RNA完整性数量显示，三种组织类型的RNA质量显着不同，其中大脑的完整性显着低于骨骼肌和心肌。研究发现，体重指数影响骨骼肌组织中RNA的完整性（M。髂腰肌）。与来自正常体重供体的样品相比，来自BMI > 25的死者的样品具有显著较低的完整性。发现正确的数据标准化部分减少了RNA质量受损所造成的影响。然而，可以得出结论，在具有低RNA完整性数目的死后组织中，检测基因表达活性的大差异可能仍然是可能的，而小的表达差异由于降解而易于误解。因此，在处理死后样本时，我们建议生成所有目标转录本的降解谱，以揭示RT-qPCR检测的检测限。
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.