Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces

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 assessmen...

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Bibliographic Details
Published in:Nucleic Acids Research 2005-01, Vol.33 (6), p.e56-e56
Main Authors: Imbeaud, Sandrine, Graudens, Esther, Boulanger, Virginie, Barlet, Xavier, Zaborski, Patrick, Eveno, Eric, Mueller, Odilo, Schroeder, Andreas, Auffray, Charles
Format: Article
Language:English
Subjects:
Cell Line
Electrophoresis, Capillary - methods
Gene Expression Profiling - standards
Humans
Life Sciences
Methods Online
Polymerase Chain Reaction
Quality Control
Reproducibility of Results
RNA - analysis
RNA - isolation & purification
RNA - metabolism
Software
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Summary: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.
ISSN:0305-1048
1362-4962
1362-4962
DOI:10.1093/nar/gni054