Hematopoietic Lineage Transcriptome Stability and Representation in PAXgene™ Collected Peripheral Blood Utilising SPIA Single-Stranded cDNA Probes for Microarray

Peripheral blood as a surrogate tissue for transcriptome profiling holds great promise for the discovery of diagnostic and prognostic disease biomarkers, particularly when target tissues of disease are not readily available. To maximize the reliability of gene expression data generated from clinical blood samples, both the sample collection and the microarray probe generation methods should be optimized to provide stabilized, reproducible and representative gene expression profiles faithfully representing the transcriptional profiles of the constituent blood cell types present in the circulation. Given the increasing innovation in this field in recent years, we investigated a combination of methodological advances in both RNA stabilisation and microarray probe generation with the goal of achieving robust, reliable and representative transcriptional profiles from whole blood. To assess the whole blood profiles, the transcriptomes of purified blood cell types were measured and compared with the global transcriptomes measured in whole blood. The results demonstrate that a combination of PAXgene™ RNA stabilising technology and single-stranded cDNA probe generation afforded by the NuGEN Ovation RNA amplification system V2™ enables an approach that yields faithful representation of specific hematopoietic cell lineage transcriptomes in whole blood without the necessity for prior sample fractionation, cell enrichment or globin reduction. Storage stability assessments of the PAXgene™ blood samples also advocate a short, fixed room temperature storage time for all PAXgene™ blood samples collected for the purposes of global transcriptional profiling in clinical studies

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Integration of macrofossil biostratigraphy and magnetostratigraphy for the Pacific Coast Upper Cretaceous (Campanian–Maastrichtian) of North America and implications for correlation with the Western Interior and Tethys

New biostratigraphic data obtained from measured stratigraphic sections of Santonian through Maastrichtian age located along the west coast of North America necessitate changes to the currently accepted chronostratigraphic framework for this region of the North Pacific biotic province. We recognize and/or define 12 molluscan zones over this interval of the Upper Cretaceous and propose revisions to the currently accepted integration of ammonite zones with global Upper Cretaceous magnetochrons. Our findings demonstrate that there was significantly more faunal interchange between the North American Pacific Coast and both the Western Interior and Gulf Coast regions of North America during the Late Cretaceous than has previously been recognized, and because of this, novel and direct biostratigraphic correlations can be made. These new faunal correlations are augmented with the magnetostratigraphic record from Pacific Coast localities to arrive at better interregional correlation for the Upper Cretaceous globally. The new integration of the global polarity time scale with the local, west coast ammonite zonation now allows better correlation between sections both within the North Pacific province (but geographically far from our study areas) as well as to sections outside of the province itself. However, we note here that previous correlations between biostratigraphy and the top and bottom of magnetochron 33r in west coast North American sections appear to have been in error due to unrecognized, modern-day normal-field overprint of originally reversed polarity in Upper Cretaceous sections. We reinterpret the position of this chron based on this new information

Comparative gene expression profiling identifies common molecular signatures of NF-κB activation in canine and human diffuse large B cell lymphoma (DLBCL)

We present the first comparison of global transcriptional changes in canine and human diffuse large B-cell lymphoma (DLBCL), with particular reference to the nuclear factor-kappa B (NF-κB) pathway. Microarray data generated from canine DLBCL and normal lymph nodes were used for differential expression, co-expression and pathway analyses, and compared with analysis of microarray data from human healthy and DLBCL lymph nodes. The comparisons at gene level were performed by mapping the probesets in canine microarrays to orthologous genes in humans and vice versa. A considerable number of differentially expressed genes between canine lymphoma and healthy lymph node samples were also found differentially expressed between human DLBCL and healthy lymph node samples. Principal component analysis using a literature-derived NF-κB target gene set mapped to orthologous canine array probesets and human array probesets clearly separated the healthy and cancer samples in both datasets. The analysis demonstrated that for both human and canine DLBCL there is activation of the NF-κB/p65 canonical pathway, indicating that canine lymphoma could be used as a model to study NF-κB-targeted therapeutics for human lymphoma. To validate this, tissue arrays were generated for canine and human NHL and immunohistochemistry was employed to assess NF-κB activation status. In addition, human and canine B-cell lymphoma lines were assessed for NF-κB activity and the effects of NF-κB inhibition

Comparison of enrichment of KEGG pathways in the co-expressed clusters of canine DLBCL and human DLBCL.

<p>The highly connected gene clusters identified in the co-expression networks of canine DLBCL and human DLBCL were analysed for enrichment of KEGG pathways using DAVID functional annotation tool. The results from the analysis of each cluster are compiled and the <i>p-values</i> of the enrichment score computed by Fisher's exact test are represented graphically as coloured icons.</p

NF-κB target genes in the differentially expressed gene set of the canine DLBCL.

<p>NF-κB target genes in the differentially expressed gene set of the canine DLBCL.</p

Hierarchical clustering of canine and human datasets using exclusively the expression levels of the NF-κB target genes (probesets).

<p>Hierarchical clustering of the canine (A) and human (B) datasets using exclusively the expression levels of the NF-κB target gene set. The samples are arranged in the columns (blue squares denote healthy and red squares denote DLBCL) and the probesets are in the rows. The dendrograms are drawn using Euclidean distances with average linkage method. (A) In the canine dataset (GSE30881), the 199 NF-κB target probesets separate the dataset into three top-level clusters. While the first and the third clusters have exclusively of DLBCL samples, the second cluster has all the healthy samples with two DLBCL samples. (B) In the human dataset (GSE12195), the 259 NF-κB target probesets separate the dataset into two top-level clusters. The first cluster has 12 samples that include all the healthy samples and two DLBCL samples, while the second cluster is solely of 43 DLBCL samples. The numbers above each column refer to sample identification numbers.</p