Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of rat Myasthenia gravis model, diaphragm, extensor digitorum longus and extraocular muscles

ABSTRACT: gene expression profile in diaphragm (DIA), extensor digitorum longus (EDL) and extraocular (EOM) muscles of rats with actively induced experimentally acquired MG (EAMG) using Affymetrix rat RAE230 gene chip. Experiment Overall Design: Total RNA was obtained with TRIzol (Invitrogen Carlsbad, CA) following the manufacturers recommended protocol. Tissues from 3 animals were combined into each RNA sample to decrease inter-subject variability. RNA pellets were cleaned by RNasey kits and resuspended at 1 Î¼g RNA/Î¼l DEPC-treated water and 5 ug was used in a reverse transcription reaction (SuperScript II; Life Technologies, Rockville, MD) to generate first strand cDNA. Double strand cDNA was synthesized and used in an in vitro transcription (IVT) reaction to generate biotinylated cRNA. Fragmented cRNA (15 ug) was used in a 300 ul hybridization cocktail containing herring sperm DNA and BSA as carrier molecules, spiked IVT controls, and buffering agents. A 200 ul aliquot of this cocktail was used for hybridization to Affymetrix rat REA230 (Santa Clara, CA) microarrays for 16 hrs at 45o C. The manufacturerâs standard post-hybridization wash, double-stain, and scanning protocols used an Affymetrix GeneChip Fluidics Station 400 and a Hewlett Packard Gene Array scanner. Experiment Overall Design: Microarray data analysis Experiment Overall Design: Raw data from microarray scans were analyzed with Affymetrix GCOS 2.0. GCOS evaluates sets of perfect match (PM) and mismatch (MM) probe sequences to obtain both hybridization signal values and present/absent calls for each transcript. Microarrays were scaled to the same target intensity and pairwise comparisons were made between experimental and control samples. Comparisons the one-sided Wilcoxonâs signed rank test to estimate âincrease/no change/ decreaseâ difference calls for each pair-wise comparison. Transcripts defined as differentially regulated met the criteria of: (a) consistent increase/decrease call across 7 out of 9 replicate comparisons, based upon Wilcoxonâs signed rank test (algorithm assesses probe pair saturation, calculates a p value and determines increase, decrease, or no change calls) and (b) the average fold difference â¥ 2.0. Data were visualized as a hierarchical dendrogram generayed on computer (Genespring software,ver 7.2; Silicon Genetics, Redwood city, CA). Annotation was done according to Affymetrix NetAffyx Gene Ontology database.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Bendi Gong

PROVIDER: E-GEOD-11465 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In muscle, reactive oxygen species (ROS) generation increases with strenuous activity, chronic unloading, and inflammatory stimuli; skeletal muscle function is very sensitive to ROS; and there are redox-sensitive signaling pathways. Using myogenic cell cultures, we asked whether hydrogen peroxide (H2O2) induces adaptive changes in skeletal muscle gene expression. H2O2 downregulated or failed to induce antioxidant or apoptotic genes in the myotubes. Instead, H2O2 changed the expression of genes for cytosolic and mitochondrial enzymes, and upregulated inflammatory mediators. Finally, H2O2 had a mostly inhibitory effect on the expression of many transcription factors. The results indicate that mild oxidative stress may induce an adaptive response in skeletal muscle without antioxidant upregulation or apoptosis. Experiment Overall Design: Cell Culture: Experiment Overall Design: C2C12 myoblasts (ATCC, Rockville, MD) were cultured in DMEM supplemented with 10% fetal calf serum at 37°C in 5% CO2. Myoblast differentiation was initiated by switching to differentiation medium (DMEM supplemented with 2% heat-inactivated horse serum) and allowed to continue for 96 hrs. Differentiated myotubes were treated for 2 hours with H2O2 (Sigma-Aldrich, St. Louis, MO) 0, 1, 10, 100, or 1000 uM in DMEM supplemented with 0.5% heat-inactivated horse serum. Experiment Overall Design: cDNA microarrays: Experiment Overall Design: Total RNA was obtained from treated C2C12 myotubes with TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's recommended protocol.Oligonucleotide microarray studies with Affymetrix Mouse Genome 430A gene chips (n=20 chips) were conducted as described earlier. Microarrays were washed and stained with a streptavidin-bound marker and scanned. Data were analyzed with Affymetrix Microarray Suite 5.0 software. Only genes with consistent absent/present calls in all four independent replicates per group were considered for further analysis. Treatment comparisons were crossed so that each H2O2 sample was compared to each control. The software uses the one-sided Wilcoxon's signed rank test to estimate increase/no change/ decrease difference calls for each pair-wise comparison. Only difference calls consistent in all pair-wise comparisons and with average changes > 1.75 were considered significant, resulting in a conservative list of genes with changed expression levels. Functional classification of genes was based on an extensive literature review.

Project description:Some of the most common neurodegenerative disorders have a strong association with aging. Curiously, these conditions tend to involve specific areas of the central and peripheral nervous system, at least initially. For example, amyotrophic lateral sclerosis preferentially targets spinal motor neurons and spares oculomotor neurons. This suggests that aging has divergent effects on brain stem and spinal cord, a factor that may explain differential susceptibility to certain neurodegenerative disorders. Therefore, for this study we asked whether aging induces a change in the evolution of the transcriptome of oculomotor nucleus (OMN) throughout the lifespan which would be different than that of the spinal cord aging. Age induced a transcriptome shift to a pattern consistent with inflammation/immune response in both OMN and spinal cord. However, the response was more dramatic in the spinal cord. On the other hand, age repressed genes coding ion channels and transporters in OMN and cytoskeletal proteins in the spinal cord. The results indicate that OMN and spinal cord transcriptomes change differently with age. The results give an initial glimpse of how tissue-specific gene expression patterns may underlie the differential susceptibility to age-related neuromuscular dysfunction. Experiment Overall Design: Total RNA was obtained with TRIzol (Invitrogen Carlsbad, CA) following the manufacturers recommended protocol. Tissues from 4 animals were combined into each RNA sample to decrease inter-subject variability. Biotinylated cRNA samples were hybridized to Affymetrix Rat Genome RA230 gene chips (n=18 chips) described previously [McMullen et al. 2004]. Microarrays were washed and stained with a streptavidin-bound marker and scanned. Data were analyzed with Affymetrix Microarray Suite 5.0 software. Only genes with consistent absent/present calls in all four independent replicates per group were considered for further analysis. Comparisons used the 6-mo transcriptome as the baseline and the one-sided Wilcoxonâ??s signed rank test to estimate â??increase/no change/ decreaseâ?? difference calls for each pair-wise comparison. Only difference calls consistent in all pair-wise comparisons and with average changes > 1.70 were considered significant, resulting in a conservative list of genes with changed expression levels. Functional classification of genes was based on an extensive literature review.

Project description:Amyotrophic lateral sclerosis (ALS) spares the ocular motor system. In this study, we tested the hypothesis that the oculomotor neurons are intrinsically protected in ALS. Using high-density cDNA microarrays, we examined the transcriptome of oculomotor nuclei and spinal cords in mice expressing a human mutant SOD1, the SOD1(G93A) ALS model, at 6 and 10 weeks of age. Comparison of gene expression profiles of these pre-symptomatic SOD1(G93A) mice showed a shift to a proapoptotic state in spinal cords, while the opposite was true in oculomotor nuclei. Seventeen members of the A, B, C and D Hox clusters increased in oculomotor nuclei from 6 to 10 weeks of age; 15 were downregulated in spinal cord. Although only the first 4 classes of a given Hox cluster (e.g., Hoxa1-4) are normally expressed in the developing hindbrain, we found differential expression of mostly the latter classes in both oculomotor nuclei and spinal cords. Also, semaphorin 3B was expressed at 28-fold greater levels in oculomotor nuclei and 61-fold less in spinal cords in 10-week old SOD1(G93A) mice compared to 6-week old mice. Semaphorins 3A and 3E were also differentially regulated. Comparison of gene expression profiles of control SOD1 mice of 6 and 10 weeks of age did not show these changes. Based on these results, we rejected our hypothesis and conclude that the oculomotor nuclei actively adapt to the ALS-inducing mutation.,Supported by NEI and ALSA. <br>Overall design<br>Oligonucleotide microarray studies using the Affymetrix system were conducted as described earlier (McMullen et al., 2004). Biotinylated cRNA was hybridized to Affymetrix Mouse Expression Set 430A GeneChips. Then, the microarrays were washed and stained with a streptavidin-bound marker, and scanned with a laser scanner. Resulting microarray data were analyzed with Affymetrix Microarray Suite 5.0 software. Only those genes with consistent absent/present calls in the three independent replicates per group were considered for further analyses. Comparisons were crossed such that each oculomotor nuclei sample was compared with each spinal cord sample at the corresponding age. The Affymetrix software uses the one-sided Wilcoxons signed rank test to estimate increase/no change/decrease difference calls and fold-changes for each pair-wise comparison. Only difference calls consistent in all pair-wise comparisons and with average changes greater than 2-fold were considered significant, resulting in a conservative list of genes with changed expression levels.

Project description:Sarcopenia is the decreased muscle mass and weakness associated with aging and a major cause of morbidity and mortality in the elderly. To what extent non-locomotive muscles are susceptible to this condition is unclear. For example, age affects laryngeal function (ventilation, airway protective reflexes, swallowing and phonation). Age-related laryngeal dysfunction may be due to effects on its intrinsic muscles that have a unique phenotype: very small, mostly fast oxidative muscle fibers. For this study, we examined how age alters the functional characteristics and gene expression profile of posterior cricoarytenoid (PCA), an intrinsic laryngeal muscle. PCA muscles from Fischer 344-Brown Norway F1 hybrid rats (6 and 30 months of age) were used for cDNA microarrays, light and electron (EM) microscopy, and in vitro contractile function. Histological analyses demonstrated a ~40% increase in mean PCA fiber size and in the number of fibers with low myosin ATPase activity. There was also evidence of ragged-red fibers, a hallmark of mitochondrial dysfunction. In turn, mitochondrial volume density, determined by EM, was significantly higher in PCA muscles at 30 months (43% vs. 21% at 6 months). In vitro function showed a decrease in velocity of unloaded shortening at 30 months. Finally, cDNA microarrays demonstrated a transcriptome shift in PCA muscle with age. Gene classes with the largest changes were: signal transduction, transcription factors, and metabolic enzymes. These data demonstrate that PCA muscles are significantly altered by age. Moreover, the observed changes in muscle fiber size, mitochondrial content and gene expression profile suggest that the PCA response to age diverges from that seen in more typical skeletal muscles. Experiment Overall Design: Total RNA was obtained with TRIzol (Invitrogen Carlsbad, CA) following the manufacturers recommended protocol. Tissues from 4 animals were combined into each RNA sample to decrease inter-subject variability. Biotinylated cRNA samples were hybridized to Affymetrix Rat Genome U34 gene chips (n=9 chips) described previously [McMullen et al. 2004]. Microarrays were washed and stained with a streptavidin-bound marker and scanned. Data were analyzed with Affymetrix Microarray Suite 5.0 software. Only genes with consistent absent/present calls in all three independent replicates per group were considered for further analysis. Comparisons used the 6-mo transcriptome as the baseline and the one-sided Wilcoxonâs signed rank test to estimate âincrease/no change/ decreaseâ difference calls for each pair-wise comparison. Only difference calls consistent in all pair-wise comparisons and with average changes > 1.70 were considered significant, resulting in a conservative list of genes with changed expression levels. Functional classification of genes was based on an extensive literature review.

Project description:The fast and constant activity of the extraocular muscles (EOMs) impose mechanical and metabolic stresses not typically seen in limb skeletal muscles. These functional properties may explain why EOMs seem to age at a faster rate than other skeletal muscles. Using high-density cDNA microarrays, this study investigated the gene expression profile of EOMs and extensor digitorum longus muscles (EDL) of Fischer 344/Brown Norway F1 hybrid rats at 6-, 18- and 30-months of age. At 6-mo, 705 genes and expressed sequence tags (ESTs) were differentially expressed in EOMs (436 up, 269 down). Overall, the EOM profile at this age was mostly consistent with the increased expression of fetal, developmental and EOM-specific myosin isoforms, enzymes involved in glycolysis and TCA cycle, and ion transporters and pumps, confirming the notion that EOM may represent a distinct muscle group (PNAS 98:12062, 2001). Interestingly, at 18-mo only 36 probes were significantly different in EOM (15 up, 21 down), most of them ESTs. However, at 30-mo EOMs had 655 differentially expressed genes and ESTs (480 up, 175 down). In this age group, the EOM expression profile reverted to a pattern similar to that found at 6-mo, with evidence of ongoing tissue remodeling and increased expression of antioxidant enzymes. These results indicate that the gene expression profile of EOM and EDL evolve differently throughout the lifespan. Experiment Overall Design: Total RNA was obtained with TRIzol (Invitrogen Carlsbad, CA) following the manufacturers recommended protocol. Tissues from 4 animals were combined into each RNA sample to decrease inter-subject variability. Biotinylated cRNA samples were hybridized to Affymetrix Rat Genome U34 gene chips (n=24 chips) described previously [McMullen et al. 2004]. Microarrays were washed and stained with a streptavidin-bound marker and scanned. Data were analyzed with Affymetrix Microarray Suite 5.0 software. Only genes with consistent absent/present calls in all four independent replicates per group were considered for further analysis. Comparisons the one-sided Wilcoxonâs signed rank test to estimate âincrease/no change/ decreaseâ difference calls for each pair-wise comparison. Only difference calls consistent in all pair-wise comparisons and with average changes > 2.00 were considered significant, resulting in a conservative list of genes with changed expression levels. Functional classification of genes was based on an extensive literature review.

Project description:Experiment design Type of experiment: Gene expression profiling analysis of S.cerevisiae W303-1a. Experimental factor: genetic variation (WT, gpr1 and gpa2 mutant strains). Number of hybridizations: 6. Single-colour labeling was performed for each sample. Sample preparation: Total RNA was prepared using FastRNA Pro Red Kit (BIO 101 Systems) according to the manufacturers' instructions. Labeling protocol 10 µg of total RNA was reverse transcribed using the SuperScript Choice system for cDNA synthesis (Life Technologies) according to the protocol recommended by Affymetrix. The oligonucleotide used for priming was 5’-ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24-3’ (Genset Oligo). Double-stranded cDNA was cleaned by phenol:chloroform extraction and the aqueous phase removed by centrifugation through Phase-lock Gel (Eppendorf). In vitro transcription was performed on 1 µg of cDNA using the Enzo BioArray High Yield RNA transcript labelling kit (Enzo Diagnostics). The cRNA was cleaned using RNAeasy clean-up columns (Qiagen). The cRNA was fragmented by heating in 40 mM Tris-acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc. Hybridization protocol 10 µg of fragmented cRNA were hybridised (45°C, 16 hours). Hybridization was controled by use of the GeneChip™ Eukaryotic Hybridization Control Kit (Affymetrix). Washing and staining was performed in a Fluidics Station 400 (Affymetrix) using the protocol EukGE-WS2v4. Measurement data, specifications and data processing Scanning was performed in an Affymetrix GeneChip scanner. Chip analysis was performed using the Affymetrix Microarray Suite v5. Changes in gene expression were assessed by looking for concordant changes between all replicates using a signed Wilcoxon rank test. The “change” p-value threshold was < 0.025 for increase and > 0.975 for decrease. Array design Microarray analysis was performed using YGS98 GeneChips™ oligonucleotide arrays (Affymetrix #900256) representing approximately 6000 ORFs. Sequences and GenBank accession numbers of all probe sets are available at the Affymetrix home page http://www.affymetrix.com/index.affx. Keywords: ordered

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Transcription profiling of rat Myasthenia gravis model, diaphragm, extensor digitorum longus and extraocular muscles

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