Differences in intensity-dependence between RNA-seq and microarrays

Understanding the differences between microarray and RNA-Seq technologies for measuring gene expression is necessary for informed design of experiments and choice of data analysis methods. Previous comparisons have come to sometimes contradictory conclusions. Reseashers from Princeton University suggest that this results from a lack of attention to the intensity-dependent nature of variation generated by the technologies.

To examine this trend, they carried out a parallel nested experiment performed simultaneously on the two technologies that systematically split variation into four stages (treatment, biological variation, library preparation and chip/lane noise), allowing a separation and comparison of the sources of variation in a well-controlled cellular system, Saccharomyces cerevisiae.


Schematic of the experiment. The Condition and Biological Replicate steps were performed irrespective of technology and these materials were then utilized in a technology-specific manner (microarrays or RNA-seq) for the Preparation and Chip/Lane steps.

With this novel dataset, the researchers demonstrate that power and accuracy are more dependent on per-gene read depth in RNA-Seq than they are on fluorescence intensity in microarrays and their quantitative PCR validations indicate that microarrays may demonstrate greater systematic bias in low-intensity genes than in RNA-seq.


Boxplots comparing the normalized and centered expression values of microarrays, RNA-Seq and qPCR of the six genes for which microarray and qPCR most disagreed. This shows that in many cases, microarray measurements were very consistent between biological, technical and chip replicates. This suggests that the problem is not variation at low-intensity microarray measurements, but rather bias.

Robinson DG, Wang JY, Storey JD. (2015) A nested parallel experiment demonstrates differences in intensity-dependence between RNA-seq and microarrays. Nucleic Acids Res [Epub ahead of print]. [article]

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