Medical Biology - Research Publications
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ItemMolecular networks involved in mouse cerebral corticogenesis and spatio-temporal regulation of Sox4 and Sox11 novel antisense transcripts revealed by transcriptome profiling(BMC, 2009)BACKGROUND: Development of the cerebral cortex requires highly specific spatio-temporal regulation of gene expression. It is proposed that transcriptome profiling of the cerebral cortex at various developmental time points or regions will reveal candidate genes and associated molecular pathways involved in cerebral corticogenesis. RESULTS: Serial analysis of gene expression (SAGE) libraries were constructed from C57BL/6 mouse cerebral cortices of age embryonic day (E) 15.5, E17.5, postnatal day (P) 1.5 and 4 to 6 months. Hierarchical clustering analysis of 561 differentially expressed transcripts showed regionalized, stage-specific and co-regulated expression profiles. SAGE expression profiles of 70 differentially expressed transcripts were validated using quantitative RT-PCR assays. Ingenuity pathway analyses of validated differentially expressed transcripts demonstrated that these transcripts possess distinctive functional properties related to various stages of cerebral corticogenesis and human neurological disorders. Genomic clustering analysis of the differentially expressed transcripts identified two highly transcribed genomic loci, Sox4 and Sox11, during embryonic cerebral corticogenesis. These loci feature unusual overlapping sense and antisense transcripts with alternative polyadenylation sites and differential expression. The Sox4 and Sox11 antisense transcripts were highly expressed in the brain compared to other mouse organs and are differentially expressed in both the proliferating and differentiating neural stem/progenitor cells and P19 (embryonal carcinoma) cells. CONCLUSIONS: We report validated gene expression profiles that have implications for understanding the associations between differentially expressed transcripts, novel targets and related disorders pertaining to cerebral corticogenesis. The study reports, for the first time, spatio-temporally regulated Sox4 and Sox11 antisense transcripts in the brain, neural stem/progenitor cells and P19 cells, suggesting they have an important role in cerebral corticogenesis and neuronal/glial cell differentiation.
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ItemIllumina WG-6 BeadChip strips should be normalized separately(BMC, 2009-11-11)BACKGROUND: Illumina Sentrix-6 Whole-Genome Expression BeadChips are relatively new microarray platforms which have been used in many microarray studies in the past few years. These Chips have a unique design in which each Chip contains six microarrays and each microarray consists of two separate physical strips, posing special challenges for precise between-array normalization of expression values. RESULTS: None of the normalization strategies proposed so far for this microarray platform allow for the possibility of systematic variation between the two strips comprising each array. That this variation can be substantial is illustrated by a data example. We demonstrate that normalizing at the strip-level rather than at the array-level can effectively remove this between-strip variation, improve the precision of gene expression measurements and discover more differentially expressed genes. The gain is substantial, yielding a 20% increase in statistical information and doubling the number of genes detected at a 5% false discovery rate. Functional analysis reveals that the extra genes found tend to have interesting biological meanings, dramatically strengthening the biological conclusions from the experiment. Strip-level normalization still outperforms array-level normalization when non-expressed probes are filtered out. CONCLUSION: Plots are proposed which demonstrate how the need for strip-level normalization relates to inconsistent intensity range variation between the strips. Strip-level normalization is recommended for the preprocessing of Illumina Sentrix-6 BeadChips whenever the intensity range is seen to be inconsistent between the strips. R code is provided to implement the recommended plots and normalization algorithms.
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ItemMicroarray background correction: maximum likelihood estimation for the normal-exponential convolution(OXFORD UNIV PRESS, 2009-04)Background correction is an important preprocessing step for microarray data that attempts to adjust the data for the ambient intensity surrounding each feature. The "normexp" method models the observed pixel intensities as the sum of 2 random variables, one normally distributed and the other exponentially distributed, representing background noise and signal, respectively. Using a saddle-point approximation, Ritchie and others (2007) found normexp to be the best background correction method for 2-color microarray data. This article develops the normexp method further by improving the estimation of the parameters. A complete mathematical development is given of the normexp model and the associated saddle-point approximation. Some subtle numerical programming issues are solved which caused the original normexp method to fail occasionally when applied to unusual data sets. A practical and reliable algorithm is developed for exact maximum likelihood estimation (MLE) using high-quality optimization software and using the saddle-point estimates as starting values. "MLE" is shown to outperform heuristic estimators proposed by other authors, both in terms of estimation accuracy and in terms of performance on real data. The saddle-point approximation is an adequate replacement in most practical situations. The performance of normexp for assessing differential expression is improved by adding a small offset to the corrected intensities.
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ItemMurine hematopoietic blast colony-forming cells and their progeny have distinctive membrane marker profiles(NATL ACAD SCIENCES, 2009-11-10)Two distinct bone marrow-derived blast colony-forming cells can generate colonies of lineage-restricted progenitor cells in agar cultures of murine bone marrow. Both cell types selectively had a Kit(+) ScaI(+) phenotype distinguishing them from most lineage-restricted progenitor cells. Multicentric blast colony-forming cells stimulated by stem cell factor plus interleukin-6 (IL-6) (BL-CFC-S) were separable from most dispersed blast colony-forming cells stimulated by Flt3 ligand and IL-6 (BL-CFC-F) using CD34 and Flt3R probes. Multicentric BL-CFC-S cofractionated with colony-forming units, spleen (CFU-S) supporting the possibility that the 2 cells may be identical. The colony populations generated by BL-CFC-S were similar in their phenotype and proliferative capacity to progenitor cells in whole bone marrow but the progeny of BL-CFC-F were skewed with an abnormally high proportion of Kit(-) Flt3R(+) cells whose clonogenic cells tended to generate only macrophage progeny. Both blast colony populations had a high percentage of GR1(+) and Mac1(+) cells but BL-CFC-F colonies also contained a significant population of B220(+) and IL-7R(+) cells relevant to the superior ability of BL-CFC-F colony cells to generate B lymphocytes and the known dependency of this process on Flt3 ligand and IL-7. The commitment events and phenotypic changes during the generation of differing progenitor cells in blast colonies can now be clonally analyzed in a convenient in vitro culture system.