Methods in molecular biology (Clifton, N.J.) 20110101
Antibody microarrays are a multiplexing technique for the analyses of hundreds of different analytes in parallel from small sample volumes of few microlitres only. With sensitivities in the picomolar to femtomolar range, they are gaining importance in proteomic analyses. These sensitivities can be obtained for complex protein samples without any pre-fractionation or signal amplification. Also, no expensive or elaborate protein depletion steps are needed. As with custom DNA-microarrays, the imple ...[more]
Project description:DNA binding profiling of endogenous HIF1A on proximal promoters in human HeLa cells exposed to 1% oxygen (hypoxia), using normoxic cells (21% oxygen) as reference.<br><br>Biological background: The Hypoxia Inducible Factor Family of transcription factors is proposed as the main orchestrator of the cellular response to hypoxia. HIFs are heterodimers of a HIF alpha and a HIF beta subunit. HIF alpha protein stability is regulated by oxygen-dependent proteasomal degradation, and hence HIFs are strongly stabilized in hypoxia.<br><br>Purpose of the study: a number of HIF1 ChIP-chip studies have been reported, employing various cell types, array platforms and HIF antibodies, and the overlap of HIF binding locations in these studies is relatively small. The aim of this study was to characterize HIF1 binding in an additional cell line (HeLa), and employing a different HIFalpha antibody.<br><br>Experimental design: We conducted a total of six hybridizations employing four biological replicates. For two biological replicates, we performed dye-swap technical replicate experiments.<br><br>Results summary: We identified 55 HIF binding locations in HeLa cells (FDR<2%). While this number is relatively low compared to previous studies, presumably due to limiting antibody sensitivity, the overlap with data from other cell lines is comparable to our HeLa data.
Project description:Although non muscle-invasive bladder cancer can be treated successfully by surgical resection, there is a high rate of recurrence, which frequently develops into an invasive form of cancer. Due to the lack of marker molecules to predict recurrence, it is currently recommended that after resection each patient is screened via cystoscopy at least twice a year. This results in a psychic burden to the patient and substantial economic costs to the health care system. Using antibody microarrays targeting 724 different cancer-related proteins, we studied protein profiles of patients with and without recurrence. The analysis revealed 255 proteins of differential abundance. Most are involved in the regulation and execution of apoptosis and cell proliferation. For prognosis, a signature of 20 proteins was determined that predicts bladder cancer recurrence with 100% sensitivity and 80% specificity. As a measure of overall accuracy, the area under the curve (AUC) value was found to be 90%. This is well within a clinically relevant window of quality and should support decision making about the stringency of surveillance or even different treatment options.
Project description:Within a study RNA samples of synovial fluid polymorphnuclear neutrophil granulocytes from 9 RA patients and of blood PMN from 4 healthy donors were analyzed for anti-apoptotic gene expression. The RNA samples were amplified and labelled red. Control samples (SigM5 cell culture) were labelled green. Each sample and control sample was co-hybrizated on a microarray with 780 inflammatory genes and 20 internal controls. Within the 19 anti-apoptotic genes tested, only the p21cip1waf1, CDKN1A, gene is significantly upregulated.
Project description:We used a mouse maternal separation model, a well-known paradigm of early adversity, to test the hypothesis that transcriptional changes in peripheral blood mononuclear cells (PBMCs) are paralleled by specific gene expression changes in three brain regions that are involved in the stress response. Furthermore, we evaluated whether gene expression profiles of PBMCs could be used to predict stress-related animal behaviours.
Project description:Phenotypic plasticity, the ability of one genotype to express different phenotypes in response to changing environmental conditions, is one of the most common phenomena characterising the living world and is not only relevant for the ecology but also for the evolution of species. Daphnia, the waterflea, is a textbook example for predator induced phenotypic plastic defences including changes in life-history, behaviour and morphology. However, the analysis of molecular mechanisms underlying these inducible defences is still in its early stages.<br><br>We exposed Daphnia magna to chemical cues of the predator Triops cancriformis to identify key processes underlying plastic defensive trait formation. D. magna is known to develop an array of morphological changes in the presence of T. cancriformis including changes of carapace morphology and cuticle hardening. To get a more comprehensive idea of this phenomenon, we studied four different genotypes originating from habitats with different predation history, reaching from predator-free to temporary habitats containing T. cancriformis.<br><br>We analysed the morphologies as well as proteomes of predator-exposed and control animals. Three genotypes showed morphological changes when the predator was present. Using a high-throughput proteomics approach, we found 294 proteins which were significantly altered in their abundance after predator exposure in a general or genotype dependant manner. Proteins connected to genotype dependant responses were related to the cuticle, protein synthesis and calcium binding whereas the yolk protein vitellogenin increased in abundance in all genotypes, indicating their involvement in a more general response. Furthermore, genotype dependant responses at the proteome level correlated well with local adaptation to Triops predation.<br><br>Altogether, our study provides new insights concerning genotype dependant and general molecular processes involved in predator-induced phenotypic plasticity in D. magna.
Project description:Male Wistar rats (6 weeks, 250g), (Charles River, Sulzfeld, Germany) were equipped with micro osmotic pumps model 2001 from Alzet (Cupertino, CA) containing 400mM PETN dissolved in DMSO or the solvent and infusion was maintained for four days at 1?l/h (10.5?g/kg/min). For direct comparison, rats were also infused with 450mM NTG (6.6?g/kg/min in ethanol or the solvent as a control). <br>After 4d rats were sacrificed by exsanguinations under Isofluran anesthesia (5% inhalant in room air), the heart was rapidly excised, placed in ice-cold Krebs-HEPES-solution (composition in g/l: 5.78 NaCl, 0.35 KCl, 0.37 CaCl2, 0.30 MgSO4, 2.1 NaHCO3, 0.14 K2HPO4, 5.21 HEPES and 2.0 D-glucose) and dissected.<br>Total RNA was isolated from the hearts. Direct labeled cDNA was synthezised and hybridized to Rat OpArray Microarray (total genom expression profiling).
Project description:We integrated metabolome and proteome profiles of the parental cell line 143B.TK- versus ρ0, including PTM analyses such as phosphorylation and ubiquitination to characterize the impact of the absence of mtDNA for the entire cell. For quantitative proteome profiling, we used a shotgun LC-MS/MS approach including the classical SILAC labeling. For comprehensive metabolome profiling, we applied a targeted LC-MS approach, based on multiple reaction monitoring (MRM).</br></br>Our study revealed that mtDNA depletion leads to a non-uniform down-regulation of the mitochondrial energy metabolism in ρ0 cells on the proteome level. Metabolites of the TCA cycle were highly dysregulated which in turn had an impact on the amino acid levels, which were up regulated. Perturbation of the mitochondrial energy metabolism could lead to an activation of the retrograde response, indicated by sets of up-regulated signaling pathways in ρ0 cells, further supported by altered phosphorylation in signaling pathways and the cytoskeleton as well as de-ubiquitination of SLC transporters.
Project description:Cross-species hybridizations of Sordaria macrospora targets on Neurospora crassa microarrays were performed with targets derived from the S. macrospora wild type undergoing sexual development (wt.sex) and the mutant strains dSmtA-1 and dSmtA-2. For each strain, two independent experiments were carried out with a dye switch in the second experiment.<br><br>The following slide/target/dye combinations were done:<br><br>slide N16-86: Cy3-target: wt.sex (1), Cy5-target: dSmtA-1 (1)<br><br>slide N16-87: Cy3-target: dSmtA-1 (2), Cy5-target: dSmtA-2(2)<br><br>slide N16-88: Cy3-target: dSmtA-2 (1), Cy5-target: wt.sex (2)
Project description:RNA was extracted form chicken jejuna at 0.33,1, 2, 4, 8, 12 and 21 days post-infection, in orally infected (1.10^5 Salmonella ) and control chickens.<br>NOTE: Normalization is performed separately! on control and infected chickens, because the objective was to investigate differences in development.