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

By S. Imbeaud, Esther Graudens, V. Boulanger, Xavier Barlet, P. Zaborski, E. Eveno, Odilo M. Mueller, Andreas Schroeder, Charles Auffray. Nucleic Acids Research, 2005

580 citations recorded by the source corpus.

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.

Subfields: Ecology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/996dcbf8144e29c4a964a3f3472bf314a4f830d5

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In Ecology

1. M. Wakem
2. M. Alsarraj
3. M. W. Pfaffl
4. Leroy Hood
5. Stephen A. Bustin
6. Marie Nguyen
7. Zhu Chen
8. Sean C. Taylor

In Molecular Biology

1. T. Nolan
2. M. W. Pfaffl
3. S. Fleige
4. R. Hands
5. Stephen A. Bustin
6. C. Prgomet
7. Sean C. Taylor
8. Marie Nguyen

Citations in the corpus

• Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5 P-type ATPase (2006, methodology): “After isolation of total RNA from midbrain cryosections (see above), we checked RNA integrity of individual human midbrains using Agilent analysis and by determining the RNA integrity number (RIN”
• RNA integrity and the effect on the real-time qRT-PCR performance. (2006, background): “Altogether, it appears that the total RNA with lower rRNA ratios is not necessarily of poor quality especially if no degradation products can observe in the electrophoretic trace (Imbeaud et al., 2005).”
• Quantification of mRNA using real-time RT-PCR (2006, methodology): “Calculating concentrations from absorbance readings is not sufficiently accurate for use with RT-qPCR technolog”
• Comprehending crystalline β-carotene accumulation by comparing engineered cell models and the natural carotenoid-rich system of citrus (2011, unknown): “It is inappropriate to compare degraded and intact samples and thus necessitates a systematic RNA integrity control prior to any qPCR analysis (Bustin and Nolan, 2004; Imbeaud et al., 2005; PérezNovo et al., 2005).”
• A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines. (2010, methodology): “By ensuring consistency in both purity and quality across all RNA samples, variability between biological replicates can be reduced (Figure 1D) (Imbeaud et al. 2005).”
• Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR (2006, background): “The examination of the RNA integrity before use in different applications enabled to compare experiments and classify the significance of final gene expression results (Imbeaud et al. 2005).”
• Systems medicine: the future of medical genomics and healthcare (2009, background): “The introduction of standards and guidelines for complete microarray workflows [43] is helping to rectify the problem; these need to cover all aspects, from RNA integrity assessment [44,45] to data analysis and reporting [46,47].”