Note: While this manuscript was under review, Carr et al. (30) independently reported the application of Taq MutS in protocols for error reduction on synthetic DNA. 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. Purity and integrity of RNA are critical elements for the overall success of RNA-based analyses, including gene expression profiling methods to assess the expression levels of thousands of genes in a single assay. Starting with low quality RNA may strongly compromise the results of downstream applications which are often labor-intensive, time-consuming and highly expensive. However, in spite of the need for standardization of RNA sample quality control, presently there is no real consensus on the best classification criteria. Conventional methods are often not sensitive enough, not specific for single-stranded RNA, and susceptible to interferences from contaminants present in the sample. For instance, when using a spectrophotometer, a ratio of absorbances at 260 and 280 nm (A 260 :A 280 ) greater than 1.8 is usually considered an acceptable indicator of RNA purity (1, 2) . However, the A 260 measurement can be compromised by the presence of genomic DNA leading to over-estimation of the actual RNA concentration. On the other hand, the A 280 measurement will estimate the presence of protein but provide no hint on possible residual organic contaminants, considered at 230 nm (3) (4) (5) . Pure RNA will have A 260 :A 230 equal to A 260 :A 280 and >1.8 (1) . A second check involves electrophoresis analysis, routinely performed using agarose gel electrophoresis, with RNA either stained with ethidium bromide (EtBr) (6) (7) (8) (9) , or the more sensitive SYBR Green dye (10) . The proportion of the ribosomal bands (28S:18S) has conventionally been viewed as the primary indicator of RNA integrity, with a ratio of 2.0 considered to be typical of 'high quality' intact RNA (1) . However, these methods are highly sample-consuming, using 0.5-2 mg total RNA and often not sensitive enough to detect slight RNA degradation. Today, microfluidic capillary electrophoresis with the Agilent 2100 bioanalyzer (Agilent Technologies, USA) has become widely used, particularly in the gene expression profiling platforms (11, 12) . It requires only a very small amount of RNA sample (as low as 200 pg), the use of a size standard during electrophoresis allows the estimation of sizes of RNA bands and the measurement appears relatively unaffected by contaminants. Integrity of *To whom correspondence should be addressed. Tel: The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org the RNA may be assessed by visualization of the 28S and 18S ribosomal RNA bands ( Figure 1A and B); an elevated threshold baseline and a decreased 28S:18S ratio, both are indicative of degradation. A broad band shows DNA contamination ( Figure 1C ). As it is apparent from a review of the literature, the standard of a 2.0 rRNA ratio is difficult to meet, especially for RNA derived from clinical samples, and it now appears that the relationship between the rRNA profile and mRNA integrity is somewhat unclear (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) . On the one hand, this may reflect unspecific damage to the RNA, including sample mishandling, postmortem degradation, massive apoptosis or necrosis, but it can reflect specific regulatory processes or external factors within the living cells. Altogether, it appears that total RNA with lower rRNA ratios is not necessarily of poor quality especially if no degradation products can be observed in the electrophoretic trace ( Figure 1D ).