Barrachina Marta, Castaño Esther, Ferrer Isidro
Institut de Neuropatologia, Servei d'Anatomia Patològica, IDIBELL-Hospital Universitari de Bellvitge, L'Hospitalet de Llobregat, Spain.
Neurochem Int. 2006 Aug;49(3):276-84. doi: 10.1016/j.neuint.2006.01.018. Epub 2006 Mar 7.
The brain tissue obtained after death is subjected to several circumstances that can affect RNA integrity. The present study has been directed to reveal possible pitfalls and to control RNA normalization in post-mortem samples in order to recognize the limitations and minimize errors when using TaqMan PCR technology. This has been carried out in samples of the frontal cortex in a series of control and diseased cases covering Parkinson's disease, dementia with Lewy bodies pure form and common form, and Alzheimer's disease. Special attention has been paid to the value of the agonal state, post-mortem delay and pH of the nervous tissue as approximate predictors of the quality of RNA, as well as to the use of the Bioanalyzer to confirm RNA preservation. In addition, since possible disease-modified mRNAs have to be normalized with ideal unaltered RNAs, TaqMan human endogenous control plates have been used to determine the endogenous control most appropriate for the study. beta-glucuronidase (GUS) and beta-actin were good endogenous controls because their expression levels showed a small variation across a representative number of control and pathological cases. RNA stability was also analysed in a paradigm mimicking cumulative delay in tissue processing. GUS mRNA levels were not modified although beta-actin mRNA levels showed degradation at 22 h. Finally, the control of RNA degradation for the normalization of genes of interest was also tested. mRNA expression levels for superoxide dismutase 1 (SOD1) and metalloproteinase domain 22 (ADAM22) were examined at several artificial post-mortem times, and their expression levels compared with those for putative controls beta-actin and GUS. In our paradigm, the expressions of SOD1 and ADAM22 were apparently not modified when normalized with beta-actin. Yet their expression levels were reduced with post-mortem delay when values were normalized with GUS. Taken together, these observations point to practical consequences in TaqMan PCR studies. Short post-mortem delays and acceptable pH of the brain are not sufficient to rule out RNA degradation. The selection of adequate endogenous controls is pivotal in the study. beta-actin and GUS are found to be good endogenous controls in these pathologies, although GUS but not beta-actin expression levels are preserved in samples with long post-mortem delay.
死后获得的脑组织会受到多种可能影响RNA完整性的情况。本研究旨在揭示可能存在的问题，并控制死后样本中的RNA标准化，以便在使用TaqMan PCR技术时认识到局限性并尽量减少误差。这一工作在一系列对照和患病病例的额叶皮质样本中进行，这些病例涵盖帕金森病、纯路易体痴呆和常见路易体痴呆以及阿尔茨海默病。特别关注濒死状态、死后延迟和神经组织pH值作为RNA质量近似预测指标的价值，以及使用生物分析仪来确认RNA的保存情况。此外由于可能发生疾病修饰的mRNA必须用理想的未改变的RNA进行标准化，因此使用TaqMan人类内参板来确定最适合该研究的内参。β-葡萄糖醛酸酶（GUS）和β-肌动蛋白是良好的内参，因为它们的表达水平在相当数量的对照和病理病例中变化较小。还在模拟组织处理累积延迟的范例中分析了RNA稳定性。尽管β-肌动蛋白mRNA水平在22小时时显示降解，但GUS mRNA水平未改变。最后还测试了用于感兴趣基因标准化的RNA降解控制。在几个模拟的死后时间点检测了超氧化物歧化酶1（SOD1）和金属蛋白酶结构域22（ADAM22）的mRNA表达水平，并将它们的表达水平与假定对照β-肌动蛋白和GUS的表达水平进行比较。在我们的范例中，用β-肌动蛋白标准化时，SOD1和ADAM22的表达明显未改变。然而，当用GUS标准化时，它们的表达水平会随着死后延迟而降低。综上所述，这些观察结果指出了TaqMan PCR研究中的实际问题。短的死后延迟和可接受的脑pH值不足以排除RNA降解。选择合适的内参在研究中至关重要。在这些病理学中，β-肌动蛋白和GUS被发现是良好的内参，尽管在死后延迟较长的样本中，GUS的表达水平得以保留，而β-肌动蛋白则不然。
