Multi-platform assessment of transcriptional profiling technologies utilizing a precise probe mapping methodology.

BACKGROUND: The arrival of RNA-seq as a high-throughput method competitive to the established microarray technologies has necessarily driven a need for comparative evaluation. To date, cross-platform comparisons of these technologies have been relatively few in number of platforms analyzed and were typically gene name annotation oriented. Here, we present a more extensive and yet precise assessment to elucidate differences and similarities in performance of numerous aspects including dynamic range, fidelity of raw signal and fold-change with sample titration, and concordance with qRT-PCR (TaqMan). To ensure that these results were not confounded by incompatible comparisons, we introduce the concept of probe mapping directed "transcript pattern". A transcript pattern identifies probe(set)s across platforms that target a common set of transcripts for a specific gene. Thus, three levels of data were examined: entire data sets, data derived from a subset of 15,442 RefSeq genes common across platforms, and data derived from the transcript pattern defined subset of 7,034 RefSeq genes. RESULTS: In general, there were substantial core similarities between all 6 platforms evaluated; but, to varying degrees, the two RNA-seq protocols outperformed three of the four microarray platforms in most categories. Notably, a fourth microarray platform, Agilent with a modified protocol, was comparable, or marginally superior, to the RNA-seq protocols within these same assessments, especially in regards to fold-change evaluation. Furthermore, these 3 platforms (Agilent and two RNA-seq methods) demonstrated over 80% fold-change concordance with the gold standard qRT-PCR (TaqMan). CONCLUSIONS: This study suggests that microarrays can perform on nearly equal footing with RNA-seq, in certain key features, specifically when the dynamic range is comparable. Furthermore, the concept of a transcript pattern has been introduced that may minimize potential confounding factors of multi-platform comparison and may be useful for similar evaluations.

P. aeruginosa infection increases morbidity in experimental cholesteatomas.

OBJECTIVES: Clinicians have long noted that infected cholesteatomas are more aggressive than uninfected ones without data to support these observations. The purpose of this study is to determine the etiological role of biofilm forming P. aeruginosa (PA) and the virulence factor, type IV pili (TFP), in the pathogenesis of experimental cholesteatomas. DESIGN: We evaluated three different PA strains and one Escherichia coli strain in cholesteatoma progression: PA14, a well-characterized wound isolate, OPPA8, an otopathogenic strain from a human cholesteatoma, OPPA8-NP, an isogenic TFP deletion mutant, and DH5α, an E. coli strain. METHODS: Cholesteatomas were induced in gerbils. We inoculated the right ear with bacteria and the left with vehicle. After 6 weeks their cholesteatomas were evaluated by micro-CT scanning. Cholesteatoma size and bone resorption were analyzed digitally. RESULTS: Results demonstrate that PA infection increases cholesteatoma size when compared to uninfected controls: OPPA8 showed an 8.9-fold increase, PA14 a 2.6-fold increase, OPPA8-NP a 1.9-fold increase, while DH5α was not increased over controls. Additionally, infected bullae showed 10 to 50% more cholesteatoma-induced bone resorption. CONCLUSIONS: In this model, PA infected cholesteatomas enlarge more rapidly and are more destructive than uninfected controls. OPPA8, the strain from a human cholesteatoma, showed the greatest enlargement and bone destruction. Additionally, we demonstrate that TFP is a virulence factor in this model because the nonpiliated isogenic mutant, OPPA8-NP, was significantly less aggressive than the wild-type OPPA8 indicating that type IV pili may be a virulence factor in this disease.