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Abstract Background Oligonucleotide arrays have become one of the most widely used high-throughput tools in biology. Due to their sensitivity to experimental conditions, normalization is a crucial step when comparing measurements from these arrays. Normalization is, however, far from a solved problem. Frequently, we encounter datasets with significant technical effects that currently available methods are not able to correct. Results We show that by a careful decomposition of probe specific amplification, hybridization and array location effects, a normalization can be performed that allows for a much improved analysis of these data. Identification of the technical sources of variation between arrays has allowed us to build statistical models that are used to estimate how the signal of individual probes is affected, based on their properties. This enables a model-based normalization that is probe-specific, in contrast with the signal intensity distribution normalization performed by many current methods. Next to this, we propose a novel way of handling background correction, enabling the use of background information to weight probes during summarization. Testing of the proposed method shows a much improved detection of differentially expressed genes over earlier proposed methods, even when tested on (experimentally tightly controlled and replicated) spike-in datasets. Conclusions When a limited number of arrays are available, or when arrays are run in different batches, technical effects have a large influence on the measured expression of genes. We show that a detailed modelling and correction of these technical effects allows for an improved analysis in these situations.

Mesenchymal progenitor cells can be differentiated in vitro into myotubes that exhibit many characteristic features of primary mammalian skeletal muscle fibers. However, in general, they do not show the functional excitation-contraction coupling or the striated sarcomere arrangement typical of mature myofibers. Epigenetic modifications have been shown to play a key role in regulating the progressional changes in transcription necessary for muscle differentiation. In this study, we demonstrate that treatment of murine C2C12 mesenchymal progenitor cells with 10 µM of the DNA methylation inhibitor 5-azacytidine (5AC) promotes myogenesis, resulting in myotubes with enhanced maturity as compared to untreated myotubes. Specifically, 5AC treatment resulted in the up-regulation of muscle genes at the myoblast stage, while at later stages nearly 50% of the 5AC-treated myotubes displayed a mature, well-defined sarcomere organization, as well as spontaneous contractions that coincided with action potentials and intracellular calcium transients. Both the percentage of striated myotubes and their contractile activity could be inhibited by 20 nM TTX, 10 µM ryanodine, and 100 µM nifedipine, suggesting that action potential-induced calcium transients are responsible for these characteristics. Our data suggest that genomic demethylation induced by 5AC overcomes an epigenetic barrier that prevents untreated C2C12 myotubes from reaching full maturity. [ABSTRACT FROM AUTHOR]

Background: Oligonucleotide arrays have become one of the most widely used high-throughput tools in biology. Due to their sensitivity to experimental conditions, normalization is a crucial step when comparing measurements from these arrays. Normalization is, however, far from a solved problem. Frequently, we encounter datasets with significant technical effects that currently available methods are not able to correct. Results: We show that by a careful decomposition of probe specific amplification, hybridization and array location effects, a normalization can be performed that allows for a much improved analysis of these data. Identification of the technical sources of variation between arrays has allowed us to build statistical models that are used to estimate how the signal of individual probes is affected, based on their properties. This enables a model-based normalization that is probe-specific, in contrast with the signal intensity distribution normalization performed by many current methods. Next to this, we propose a novel way of handling background correction, enabling the use of background information to weight probes during summarization. Testing of the proposed method shows a much improved detection of differentially expressed genes over earlier proposed methods, even when tested on (experimentally tightly controlled and replicated) spike-in datasets. Conclusions: When a limited number of arrays are available, or when arrays are run in different batches, technical effects have a large influence on the measured expression of genes. We show that a detailed modelling and correction of these technical effects allows for an improved analysis in these situations. [ABSTRACT FROM AUTHOR]

Abstract: Wnt signaling has been implicated in regulating bone formation by controlling osteoblast proliferation and function. Although stabilization of β-catenin by Wnt has been shown to increase alkaline phosphatase expression and osteoblast differentiation, the precise role of Wnt signaling during the process of osteoblast differentiation is largely unknown. In this study, we used microarray technology to investigate expression regulation of Wnt signaling components during in vitro osteoblast differentiation. Expression was analyzed during bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation of murine C2C12 and MC3T3 cells and data were compared with expression in BMP2-treated NIH3T3 fibroblasts. During osteoblast differentiation, particularly strong expression regulation of the Wnt antagonists Sfrp2 (secreted frizzled related protein 2) and Wif1 (Wnt inhibitory factor 1) was observed in the late phase of differentiation. In situ expression analysis in murine tail vertebrae supported Wif1 expression during late phase bone cell differentiation, since Wif1 was found to be expressed in vivo in trabecular, but not in cortical bone. We further analyzed the effects of continuous activation of Wnt signaling by lithium chloride and observed that osteoblast differentiation was reduced, as measured by expression of osteoblast marker genes encoding alkaline phosphatase, osteocalcin, and osterix, as well as by the amount of calcium release. Taken together, our data indicate that endogenous expression of Wnt antagonists by osteoblasts provides a negative Wnt feedback loop which is essential in controlling osteoblast maturation. [Copyright &y& Elsevier]