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 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.
Imbeaud
Sandrine
S
Array s/IMAGE, Genexpress, Functional Genomics and Systems Biology for Health LGN-UMR 7091, CNRS and Pierre and Marie Curie University of Paris 6, 7, rue Guy Môquet, 94801 Villejuif, France. imbeaud@vjf.cnrs.fr
Graudens
Esther
E
Boulanger
Virginie
V
Barlet
Xavier
X
Zaborski
Patrick
P
Eveno
Eric
E
Mueller
Odilo
O
Schroeder
Andreas
A
Auffray
Charles
C
D002460:Cell Line; D019075:Electrophoresis, Capillary / Q000379:methods*; D020869:Gene Expression Profiling / Q000592:standards*; D006801:Humans; D016133:Polymerase Chain Reaction; D011786:Quality Control; D012313:RNA / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results; D012984:Software
D012313:RNA