Evaluating RNA status for RT-PCR in extracts of postmortem human brain tissue
Evaluating RNA status for RT-PCR in extracts of postmortem human brain tissue
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DOI：
10.2144/04364st03
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发表时间：
2004-04-01
期刊：
影响因子：
2.7
通讯作者：
Yolken, RH
中科院分区：
文献类型：
--
作者：
Miller, CL;Diglisic, S;Yolken, RH
从代表一系列死后条件的 105 个个体死后人脑样本中提取总 RNA。为了改进目前用于筛选 RNA 质量的参数，将 Agilent Bioanalyzer 2100 生成的电泳图谱与每个 RNA 样品中四个管家基因的随机六聚体引物逆转录实时 PCR 的平均得分进行了比较。研究发现核糖体比率（28S 与 18S）与看家基因评分无关（r = -0.06；P = 0.50），并且该比率没有可用于有效对 RNA 降解进行分类的阈值。尽管管家基因评分与电泳图中对应于中等到高分子量完整 mRNA 的面积百分比显着相关（r = 0.41；P = 0.0001），但最佳判别器被确定为 tRNA 和 18S rRNA 基线中 18S 峰高与最高峰的比率。应用比率下限 2.12 可以筛选出持家基因得分最低的样本，而不排除质量较好的样本。该测量值代表了 28S 与 18S 比率的显着改善，事实证明，28S 与 18S 比率是随机六聚体引物逆转录 PCR 中使用的 mRNA 状态的误导性指标。
Total RNA was extracted from 105 individual postmortem human brain samples representing a range of postmortem conditions. To improve upon parameters currently used to screen for RNA quality, electropherogram patterns generated by the Agilent Bioanalyzer 2100 were compared to the average score in random hexamer-primed reverse transcription real-time PCR for four housekeeping genes in each RNA sample. The ribosomal ratio (28S to 18S) was found to be unrelated to the housekeeping gene score (r = -0.06; P = 0.50), and there was no threshold value in the ratio that could be applied to effectively categorize the RNA degradation. Although the housekeeping gene score correlated significantly with the percentage of area in the electropherogram corresponding to moderate to high molecular weight intact mRNA (r = 0.41; P = 0.0001), the best discriminator was determined to be the ratio of the 18S peak height to the highest peak in the tRNA to 18S rRNA baseline. Applying a lower boundary of 2.12 for the ratio allowed for the screening out of samples with the lowest housekeeping gene scores without excluding better-quality samples. This measure represents a marked improvement over the 28S to 18S ratio, which proved to be a misleading indicator of the state of the mRNA for use in random hexamer-primed reverse transcription PCR.