精神分裂症脑队列中参考基因表达的选择。
Selection of reference gene expression in a schizophrenia brain cohort.
机构信息
Schizophrenia Research Institute, Sydney, New South Wales, Australia.
出版信息
Aust N Z J Psychiatry. 2010 Jan;44(1):59-70. doi: 10.3109/00048670903393662.
OBJECTIVE
In order to conduct postmortem human brain research into the neuropatho-logical basis of schizophrenia, it is critical to establish cohorts that are well-characterized and well-matched. The aim of the present study was therefore to determine if specimen characteristics including: diagnosis, age, postmortem interval (PMI), brain acidity (pH), and/or the agonal state of the subject at death related to RNA quality, and to determine the most appropriate reference gene mRNAs.
METHODS
A matched cohort was selected of 74 subjects (schizophrenia/schizoaffective disorder, n = 37; controls, n = 37). Middle frontal gyrus tissue was pulverized, tissue pH was measured, RNA isolated for cDNA from each case, and RNA integrity number (RIN) measurements were assessed. Using quantitative reverse transcription-polymerase chain reaction, nine housekeeper genes were measured and a geomean calculated per case in each diagnostic group.
RESULTS
The RINs were very good (mean = 7.3) and all nine housekeeper control genes were significantly correlated with RIN. Seven of nine housekeeper genes were also correlated with pH; two clinical variables, agonal state and duration of illness, did have an effect on some control mRNAs. No major impact of PMI or freezer time on housekeeper mRNAs was detected. The results show that people with schizophrenia had significantly less PPIA and SDHA mRNA and tended to have less GUSB and B2M mRNA, suggesting that these control genes may not be good candidates for normalization.
CONCLUSIONS
In the present cohort <10% variability in RINs was detected and the diagnostic groups were well matched overall. The cohort was adequately powered (0.80-0.90) to detect mRNA differences (25%) due to disease. The study suggests that multiple factors should be considered in mRNA expression studies of human brain tissues. When schizophrenia cases are adequately matched to control cases subtle differences in gene expression can be reliably detected.
目的
为了对精神分裂症的神经病理学基础进行死后人脑研究，建立特征良好且匹配良好的队列至关重要。因此，本研究旨在确定标本特征，包括：诊断、年龄、死后间隔（PMI）、脑酸度（pH）和/或死亡时受试者的濒死状态与 RNA 质量的关系，并确定最合适的参考基因 mRNA。
方法
选择了一个匹配的队列，共 74 例（精神分裂症/分裂情感障碍，n=37；对照组，n=37）。对中额回组织进行粉碎，测量组织 pH 值，从每个病例中提取 cDNA 的 RNA，并评估 RNA 完整性数（RIN）测量值。使用定量逆转录聚合酶链反应，测量了 9 种管家基因，并计算了每个诊断组中每个病例的几何平均值。
结果
RIN 非常好（平均值=7.3），所有 9 种管家基因控制基因均与 RIN 显著相关。七种管家基因与 pH 值相关；两个临床变量，濒死状态和疾病持续时间，对某些对照 mRNAs 有影响。未检测到 PMI 或冷冻时间对管家基因 mRNAs 的主要影响。结果表明，精神分裂症患者的 PPIA 和 SDHA mRNA 明显减少，GUSB 和 B2M mRNA 减少的趋势明显，表明这些对照基因可能不是很好的归一化候选基因。
结论
在本队列中，RIN 的变异性<10%，总体上诊断组匹配良好。该队列有足够的效力（0.80-0.90）来检测由于疾病导致的 mRNA 差异（25%）。该研究表明，在人脑组织的 mRNA 表达研究中应考虑多种因素。当精神分裂症病例与对照组病例充分匹配时，可以可靠地检测到基因表达的细微差异。
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