A systematic spatial bias in microarray hybridizations caused by probe spot position-dependent variability in lateral diffusion
ABSTRACT: The hybridization of nucleic acid targets with surface-immobilized probes is a widely used assay format for high-throughput detection of many parallel targetsin medical and biological research. Though commonly applied, microarray technology still suffers limitations arising from problems of data robustness and reproducibility across platforms, stemming in part from an incomplete understanding of the complex processes governing surface hybridization behavior. It has been observed that there are non-random spatial variations within individual microarray hybridizations, but the causative mechanisms of positional bias remain largely unexplained. This study identifies a symptomatic spatial bias in surface hybridization signal intensitywith systematically increased signal intensities of spots located at the boundaries of the spotted areas of the microarray slide and characterizes the underlying mechanistic principle of this bias using a simplified block array format. Experimentally-derived hybridization dynamics are compared with a mathematical modeling analysis, which together showthat the driver of the spatial bias is a position-dependent variation in lateral diffusion. Numerical simulations employing a diffusion-based model are used to demonstrate the strong influence of microarray well geometry on this spatial bias and to determine optimal conditions for which this bias can be minimized or eliminated, resulting in increased uniformity of microarray hybridization. A simplified square array of 900 identical spotted probes was hybridized using different target concentrations and different time periods to analyze systematically spatial biases in microarray hybridizations.
Project description:The hybridization of nucleic acid targets with surface-immobilized probes is a widely used assay format for high-throughput detection of many parallel targetsin medical and biological research. Though commonly applied, microarray technology still suffers limitations arising from problems of data robustness and reproducibility across platforms, stemming in part from an incomplete understanding of the complex processes governing surface hybridization behavior. It has been observed that there are non-random spatial variations within individual microarray hybridizations, but the causative mechanisms of positional bias remain largely unexplained. This study identifies a symptomatic spatial bias in surface hybridization signal intensitywith systematically increased signal intensities of spots located at the boundaries of the spotted areas of the microarray slide and characterizes the underlying mechanistic principle of this bias using a simplified block array format. Experimentally-derived hybridization dynamics are compared with a mathematical modeling analysis, which together showthat the driver of the spatial bias is a position-dependent variation in lateral diffusion. Numerical simulations employing a diffusion-based model are used to demonstrate the strong influence of microarray well geometry on this spatial bias and to determine optimal conditions for which this bias can be minimized or eliminated, resulting in increased uniformity of microarray hybridization. Overall design: A simplified square array of 900 identical spotted probes was hybridized using different target concentrations and different time periods to analyze systematically spatial biases in microarray hybridizations. Whole genome oligonucleotide microarray for Protochlamydia amoebophila. A simplified square array of 5040 identical spotted probes was hybridized using 300 ng of Cy3 labeled RNA to analyze systematically spatial biases in microarray hybridizations.
Project description:Genome-wide microarray analysis of the effects of swim-training on caudal fin development in zebrafish larvae. Zebrafish were subjected to swim-training from 5 days post fertilization (dpf) until 10 dpf. Subsequently, we performed a genome-wide microarray analysis on the caudal fins of control and trained fish at 10 dpf. The goal of the project was to investigate the effects of swim-training on the gene expression level during caudal fin development in zebrafish larvae. Two-condition experiment: control vs trained fish. RNA was isolated from pooled caudal fins of 15 control fish (in duplo: pooled control samples (C2 and C3)) and of 15 trained fish (in duplo: pooled trained samples( T2 and T3)). Subsequently, each pooled RNA sample of control and trained caudal fins was labeled with Cy3 and Cy5 in order to correct for dye bias. We included a technical replicate of the labeled C2 and T2 samples.
Project description:We characterized the bacterial diversity of chlorinated drinking water from three surface water treatment plants supplying the city of Paris, France. For this purpose, we used serial analysis of V6 ribosomal sequence tag (SARST-V6) to produce concatemers of PCR-amplified ribosomal sequence tags (RSTs) from the V6 hypervariable region of the 16S rRNA gene for sequence analysis. Using SARST-V6, we obtained bacterial profiles for each drinking water sample, demonstrating a strikingly high degree of biodiversity dominated by a large collection of low-abundance phylotypes. In all water samples, between 57.2-77.4% of the sequences obtained indicated bacteria belonging to the Proteobacteria phylum. Full-length 16S rDNA sequences were also generated for each sample, and comparison of the RSTs with these sequences confirmed the accurate assignment for several abundant bacterial phyla identified by SARST-V6 analysis, including members of unclassified bacteria, which account for 6.3-36.5% of all V6 sequences. These results suggest that these bacteria may correspond to a common group adapted to drinking water systems. The V6 primers used were subsequently evaluated with a computer algorithm to assess their hybridization efficiency. Potential errors associated with primer-template mismatches and their impacts on taxonomic group detection were investigated. The biodiversity present in all three drinking water samples suggests that the bacterial load of the drinking water leaving treatment plants may play an important role in determining the downstream community dynamics of water distribution networks. 3 different drinking water samples (Orly, Ivry, Joinville drinking water sample)
Project description:Obesity and its consequences on cardiometabolic health have been associated to low-grade inflammation. The most diverse source of dietary anti-inflammatory compounds is polyphenols and especially anthocyanins, which are major polyphenols in bilberries (Vaccinium myrtillus). We investigated the effects of a bilberry-rich diet on glucose and lipid metabolism, inflammation and gene expression profile in peripheral blood mononuclear cells (PBMCs) in subjects with overweight and other features of the metabolic syndrome. The study was a randomized, controlled clinical intervention using 2-arm parallel group design. The participants in the bilberry group (BB, n = 15) consumed bilberries or berry products equivalent of 400 g fresh bilberries daily for 8 weeks, while the participants in the control group (C, n = 12) were asked to maintain their habitual diet. The microarray profiling was done from 3 subjects in the BB group and further QPCR expression analyses from all subjects in both groups at the start and end of the intervention (weeks 0 and 8). From 50 differentially expressed transcripts (P<0.005), five candidate genes; WDSUB1, COX7B, RGS18, DAPP1 and TICAM1, were randomly selected for QPCR analyses from PBMCs in both groups. To further explore the interplay of dietary change and activated pathways in PBMCs, 11 additional genes were selected for QPCR. The selected transcripts were from the LPB, RIPK-1, Ly96 (MD2), CD19, MMD, TNFRSF12A, CD72, CCR2, IL17RC, IL17R and MAP3K7IP2 genes. Our results indicate that regular bilberry intake may reduce endotoxemia and chronic inflammation, the latter especially by directing the immunity away from overactive innate cell mediated responsiveness. Bilberry consumption may decrease cardiovascular and metabolic risk in the long term. The microarray profiling was done in PBMCs from 3 subjects in BB group and further QPCR expression analyses in PBMCs from 15 subjects in the BB group and 12 subjects in the C group. For QPCR expression analyses; Time point (biological replicate): 0 wk: baseline PBMCs1 - 15, baseline PBMCc1- 12 Time point (biological replicate): 8 wk: bilberry PBMCs1 - 15, bilberry PBMCc1 - 12 Non-normalized data with triplicate samples (technical replicate) of each bilogical replicates (replicate1-3)
Project description:Embryogenesis in rice shows a non-stereotypic cell division pattern, the formation of dorsal-ventral polarity, and endogenous initiation of the radicle, which differs from most dicotyledonous plants. To reveal the transcriptional features associated with developmental events during early embryogenesis in rice, we obtained transcriptome data, including the spatial and temporal data sets, using microarray experiments combined with a laser microdissection. We could predict the spatial expression foci of each expressed gene in the globular embryo, which revealed that the expression bias along the apical-basal and dorsal-ventral axes was correlated with temporal changes in expression levels during early embryogenesis. The spatial expression patterns of transcripts was suggestive of a potential contribution of phytohormone-related genes to early embryogenesis and was a characteristic feature of transcription factor genes. We also analyzed the relationship between expression sites of the genes in the globular embryo and those in embryonic organs at later stages. Based on in silico prediction of gene expression sites, we developed potential marker genes expressed in specific domains of the early embryo. The results and database will provide a framework for spatio-temporal gene expression in rice embryogenesis and increase our understanding of developmental diversity of plant embryogenesis. Overall design: We designed microarray experiments for two types of dataset; i.e., spatial and temporal datasets. For temporal datasets, the entire embryonic tissues at 2, 3 and 4 DAP were collected using laser microdissection. For spatial datasets, 3-DAP embryos were separated into half parts of the embryo along the A-B and D-V axis using LM, which resulted in four tissues derived from the apical, basal, dorsal and ventral regions of the embryo. In addition to the spatial datasets for early embryos, we obtained sub-spatial datasets from tissues of 7-DAP embryos. Embryo tissues closely associated with four embryonic organs; namely, the shoot, scutellem, root (radicle), and epiblast/coleorhizae, were separated and collected by LM. These 11 samples were obtained in triplicate and used to perform the 44K rice microarray.
Project description:Background: Freshwater planarians are well known for their regenerative abilities. Less well known is how planarians maintain spatial patterning in long-lived adult animals or how they re-pattern tissues during regeneration. HOX genes are good candidates to regulate planarian spatial patterning, yet the full complement or genomic clustering of planarian HOX genes has not yet been described, primarily because only a few have been detectable by in situ hybridization, and none have given morphological phenotypes when knocked down by RNAi. Results: Because the planarian Schmidtea mediterranea (S. med) is unsegmented, appendage-less, and morphologically simple, it has been proposed that it may have a simplified HOX gene complement. Here we argue against this hypothesis and show that S. med has a total of 13 HOX genes, which represent homologs to all major axial categories, and can be detected by whole-mount in situ hybridization using a highly-sensitive method. In addition, we show that planarian HOX genes do not cluster in the genome, yet 5/13 have retained aspects of axially-restricted expression. Finally, we confirm HOX gene axial expression by RNA-deep-sequencing 6 anterior-to-posterior “zones” of the animal, which we provide as a dataset to the community to discover other axially-restricted transcripts. Conclusions: Freshwater planarians have an unappreciated HOX gene complexity, with all major axial categories represented. However, we conclude based on adult expression patterns that planarians have a derived body plan and their asexual lifestyle may have allowed for large changes in HOX expression from the last common ancestor between arthropods, flatworms, and vertebrates. Using our in situ method and axial zone RNAseq data, it should be possible to further understand the pathways that pattern the anterior-posterior axis of adult planarians. Overall design: 10 animals were immobilized on peltier coolers and carefully amputated into 6 Anterior-Posterior zones, as illustrated in Figure 2 of the manuscript. RNA was extracted from each zone, and deep sequenced to a depth >52 million reads.
Project description:A phylogenetic microarray targeting 66 families described in the human gut microbiota has been developped aud used to monitor the gut microbiota's structure and diversity. The microarray format provided by Agilent and used in this study is 8x15K. A study with a total of 4 chips was realized. Arrays 1 and 2: Hybridization with 100ng of labelled 16S rRNA gene amplicons from a mock community sample and 250ng of labelled 16S rRNA gene amplicons from 1 faecal sample. Each Agilent-030618 array probe (4441) was synthetized in three replicates. Arrays 3 and 4: Hybridization with 250ng of labelled 16S rRNA gene amplicons from 2 faecal samples. Each Agilent-40558 array probe (4441) was synthetized in three replicates.
Project description:Neves2008 - Role of cell shape and size in controlling intracellular signalling
The role of cell shape and size in the flow of spatial information from the cell surface receptor to downstream components within the cell has been studied on the β-adrenergic receptor to MAPK-signalling network.
This model is described in the article:
Cell shape and negative links in regulatory motifs together control spatial information flow in signaling networks.
Neves SR, Tsokas P, Sarkar A, Grace EA, Rangamani P, Taubenfeld SM, Alberini CM, Schaff JC, Blitzer RD, Moraru II, Iyengar R
Cell. 2008, 133(4):666-680
The role of cell size and shape in controlling local intracellular signaling reactions, and how this spatial information originates and is propagated, is not well understood. We have used partial differential equations to model the flow of spatial information from the beta-adrenergic receptor to MAPK1,2 through the cAMP/PKA/B-Raf/MAPK1,2 network in neurons using real geometries. The numerical simulations indicated that cell shape controls the dynamics of local biochemical activity of signal-modulated negative regulators, such as phosphodiesterases and protein phosphatases within regulatory loops to determine the size of microdomains of activated signaling components. The model prediction that negative regulators control the flow of spatial information to downstream components was verified experimentally in rat hippocampal slices. These results suggest a mechanism by which cellular geometry, the presence of regulatory loops with negative regulators, and key reaction rates all together control spatial information transfer and microdomain characteristics within cells.
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