Lipska Barbara K, Deep-Soboslay Amy, Weickert Cynthia Shannon, Hyde Thomas M, Martin Catherine E, Herman Mary M, Kleinman Joel E
Clinical Brain Disorders Branch, Division of Intramural Research Programs, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892-1385, USA.
Biol Psychiatry. 2006 Sep 15;60(6):650-8. doi: 10.1016/j.biopsych.2006.06.019.
Studies of postmortem human brain are important for investigating underlying pathogenic molecular mechanisms of neuropsychiatric disorders. They are, however, confounded by pre- and postmortem factors. The purpose of this study was to identify sources of variation that will enable a better design of gene expression studies and higher reliability of gene expression data.
We assessed the contribution of multiple variables to messenger RNA (mRNA) expression of reference (housekeeping) genes measured by reverse transcriptase-polymerase chain reaction (RT-PCR) by multiple regression analysis in a large number (N = 143) of autopsy samples from the hippocampus and white and grey matter of the dorsolateral prefrontal cortex (DLPFC) of patients with schizophrenia and normal control subjects.
The strongest predictor of gene expression was total RNA quality. Other significant factors included pH, postmortem interval, age and the duration of the agonal state, but the importance of these factors depended on transcript measured, brain region analyzed, and diagnosis. The quality of RNA obtained from the DLPFC white matter was also adversely affected by smoking.
Our results show that normalization of expression data of target genes with a geometric mean of multiple housekeeping genes should be used to control for differences in RNA quality between samples. The results also suggest that accurate assessment of other confounding factors and their inclusion as regressors in the analysis is critical for obtaining reliable and accurate quantification of mRNA expression.
对人类大脑进行尸检研究对于探究神经精神疾病潜在的致病分子机制非常重要。然而，这些研究受到生前和死后多种因素的干扰。本研究的目的是确定变异来源，以便更好地设计基因表达研究并提高基因表达数据的可靠性。
我们通过多元回归分析，评估了多个变量对通过逆转录聚合酶链反应（RT-PCR）测量的参考（管家）基因信使核糖核酸（mRNA）表达的贡献，分析对象为大量（N = 143）来自精神分裂症患者及正常对照者海马体、背外侧前额叶皮质（DLPFC）白质和灰质的尸检样本。
基因表达的最强预测因素是总RNA质量。其他显著因素包括pH值、死后间隔时间、年龄和濒死期持续时间，但这些因素的重要性取决于所测量的转录本、分析的脑区以及诊断情况。从DLPFC白质获取的RNA质量也受到吸烟的不利影响。
我们的结果表明，应使用多个管家基因几何平均值对目标基因表达数据进行标准化，以控制样本间RNA质量的差异。结果还表明，准确评估其他混杂因素并将其作为回归变量纳入分析，对于获得可靠且准确的mRNA表达定量至关重要。
