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Abstract Background The fossil record has suggested that clade growth may differ in marine and terrestrial taxa, supporting equilibrial models in the former and expansionist models in the latter. However, incomplete sampling may bias findings based on fossil data alone. To attempt to correct for such bias, we assemble phylogenetic supertrees on one of the oldest clades of insects, the Odonatoidea (dragonflies, damselflies and their extinct relatives), using MRP and MRC. We use the trees to determine when, and in what clades, changes in taxonomic richness have occurred. We then test whether equilibrial or expansionist models are supported by fossil data alone, and whether findings differ when phylogenetic information is used to infer gaps in the fossil record. Results There is broad agreement in family-level relationships between both supertrees, though with some uncertainty along the backbone of the tree regarding dragonflies (Anisoptera). "Anisozygoptera" are shown to be paraphyletic when fossil information is taken into account. In both trees, decreases in net diversification are associated with species-poor extant families (Neopetaliidae, Hemiphlebiidae), and an upshift is associated with Calopterygidae + Polythoridae. When ghost ranges are inferred from the fossil record, many families are shown to have much earlier origination dates. In a phylogenetic context, the number of family-level lineages is shown to be up to twice as high as the fossil record alone suggests through the Cretaceous and Cenozoic, and a logistic increase in richness is detected in contrast to an exponential increase indicated by fossils alone. Conclusions Our analysis supports the notion that taxa, which appear to have diversified exponentially using fossil data, may in fact have diversified more logistically. This in turn suggests that one of the major apparent differences between the marine and terrestrial fossil record may simply be an artifact of incomplete sampling. Our results also support previous notions that adult colouration plays an important role in odonate radiation, and that Anisozygoptera should be grouped in a single inclusive taxon with Anisoptera, separate from Zygoptera.

Abstract Background The challenge of gene expression studies is to reliably quantify levels of transcripts, but this is hindered by a number of factors including sample availability, handling and storage. The PAXgene™ Blood RNA System includes a stabilizing additive in a plastic evacuated tube, but requires 2.5 mL blood, which makes routine implementation impractical for paediatric use. The aim of this study was to modify the PAXgene™ Blood RNA System kit protocol for application to small, sick chidren, without compromising RNA integrity, and subsequently to perform quantitative analysis of ICAM and interleukin-6 gene expression. Aliquots of 0.86 mL PAXgene™ reagent were put into microtubes and 0.3 mL whole blood added to maintain the same recommended proportions as in the PAXgene™ evacuated tube system. RNA quality was assessed using the Agilent BioAnalyser 2100 and an in-house TaqMan™ assay which measures GAPDH transcript integrity by determining 3' to 5' ratios. qPCR analysis was performed on an additional panel of 7 housekeeping genes. Three reference genes (HPRT1, YWHAZ and GAPDH) were identified using the GeNORM algorithm, which were subsequently used to normalising target gene expression levels. ICAM-1 and IL-6 gene expression were measured in 87 Malawian children with invasive pneumococcal disease. Results Total RNA yield was between 1,114 and 2,950 ng and the BioAnalyser 2100 demonstrated discernible 18s and 28s bands. The cycle threshold values obtained for the seven housekeeping genes were between 15 and 30 and showed good consistency. Median relative ICAM and IL-6 gene expression were significantly reduced in non-survivors compared to survivors (ICAM: 3.56 vs 4.41, p = 0.04, and IL-6: 2.16 vs 6.73, p = 0.02). Conclusion We have successfully modified the PAXgene™ blood collection system for use in small children and demonstrated preservation of RNA integrity and successful quantitative real-time PCR analysis.

Abstract Background Used alone, the MAS5.0 algorithm for generating expression summaries has been criticized for high False Positive rates resulting from exaggerated variance at low intensities. Results Here we show, with replicated cell line data, that, when used alongside detection calls, MAS5 can be both selective and sensitive. A set of differentially expressed transcripts were identified that were found to be changing by MAS5, but unchanging by RMA and GCRMA. Subsequent analysis by real time PCR confirmed these changes. In addition, with the Latin square datasets often used to assess expression summary algorithms, filtered MAS5.0 was found to have performance approaching that of its peers. Conclusion When used alongside detection calls, MAS5 is a sensitive and selective algorithm for identifying differentially expressed genes.