Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces.
Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces.
复制标题
DOI：
10.1093/nar/gni054
复制
发表时间：
2005-03-30
影响因子：
14.9
通讯作者：
Auffray C
中科院分区：
文献类型：
--
作者：
Imbeaud S;Graudens E;Boulanger V;Barlet X;Zaborski P;Eveno E;Mueller O;Schroeder A;Auffray C
虽然普遍认为完整RNA构成转录稳态的最佳代表，但在基因表达分析之前没有金标准来定义RNA质量。在这份报告中，我们评估了RNA质量评估的传统方法的可靠性，包括紫外光谱和28 S：18 S面积比，并证明了它们的不一致性。然后，我们使用了两个新的免费提供的分类器，降解仪和RIN系统，产生用户独立的RNA质量指标，基于微毛细管电泳痕迹分析。两者都提供了信息量很大和很有价值的数据，发现结果高度相关，而RIN系统提供了更可靠的数据。通过Q-PCR测试用于评估基因表达差异的RNA质量度量的相关性，揭示了不同质量的RNA样品中基因的相对表达的显著下降，而发现相似的、甚至差的完整性的样品具有高度可比性。我们讨论了这些意见的后果，以尽量减少人为检测的假阳性和阴性的差异表达，由于RNA完整性的差异，并提出了一个计划，制定一个标准的操作程序，可选注册的RNA完整性指标在公共知识库的基因表达数据。
While it is universally accepted that intact RNA constitutes the best representation of the steady-state of transcription, there is no gold standard to define RNA quality prior to gene expression analysis. In this report, we evaluated the reliability of conventional methods for RNA quality assessment including UV spectroscopy and 28S:18S area ratios, and demonstrated their inconsistency. We then used two new freely available classifiers, the Degradometer and RIN systems, to produce user-independent RNA quality metrics, based on analysis of microcapillary electrophoresis traces. Both provided highly informative and valuable data and the results were found highly correlated, while the RIN system gave more reliable data. The relevance of the RNA quality metrics for assessment of gene expression differences was tested by Q-PCR, revealing a significant decline of the relative expression of genes in RNA samples of disparate quality, while samples of similar, even poor integrity were found highly comparable. We discuss the consequences of these observations to minimize artifactual detection of false positive and negative differential expression due to RNA integrity differences, and propose a scheme for the development of a standard operational procedure, with optional registration of RNA integrity metrics in public repositories of gene expression data.
登录
查看更多内容
影响因子：
14.9
作者：
Imbeaud S;Graudens E;Boulanger V;Barlet X;Zaborski P;Eveno E;Mueller O;Schroeder A;Auffray C
通讯作者：
Auffray C
影响因子：
2.9
作者：
LEHRACH, H;DIAMOND, D;BOEDTKER, H
通讯作者：
BOEDTKER, H
影响因子：
6.6
作者：
Margan, SH;Handelsman, DJ;Dong, QH
通讯作者：
Dong, QH
影响因子：
5.3
作者：
MORI, N;MIZUNO, D;GOTO, S
通讯作者：
GOTO, S
影响因子：
6.8
作者：
Wex, T;Treiber, G;Malfertheiner, P
通讯作者：
Malfertheiner, P