Project description:In this study we aimed to obtain gene expression data during development of Drosophila melanogaster. Therefore 10 critical stages from egg to adult fly were chosen. Specifically the purpose of the project was to study gene expression in the under-replicated regions of polytene chromosomes that were discovered earlier (see GSE2551). Biological samples from ten developmental stages were collected: 0-1 hour embryos, 5-6 hours embryos, 11-12 hours embryos, 1st instar larvae 0-1 hour after hatching, 2nd instar larvae after molt, 3rd instar larvae after molt, 0-1 hour prepupae, 13-14 hours pupae, adult males and adult females. 0-1h embryos demonstrate maternally expressed genes. 5-6h embryos represent early zygotic expression. 11-12h embryos represent organogenesis of larva. 1st instar larvae are freshly hatched completely formed larvae. 2nd instar larvae were collected after first molt. 3rd instar larvae were collected after second molt. 0-1h prepupae represent early metamorphosis. 13-14h pupae represent organogenesis of adult fly. Adult males and females collected separately represent differences between sexes. Embryos were collected as follows: flies were allowed to lay eggs for 45 min on the agar/apple juice medium with some yeast. Embryos were either collected immediately and frozen at -80°C or allowed to develop at 25°C to the stage of interest and then collected and frozen. 1st instar larvae were collected right after hatching. 2nd and 3rd larvae were collected after corresponding molts, 24 hours and 48 hours after hatching. 0-1 hour prepupae were picked each hour from the tube walls and either frozen at -80°C immediately or allowed to develop at 25°C to for 13 hours to obtain 13-14 hours pupae. Adult males and females were collected twice a day, kept for 3 days at 25°C and then separated and frozen at -80°C. The females collected in this way were mated. Total RNA isolation from the frozen samples was performed with TRIzol reagent (Invitrogen, #155-96-011). RNAs from ten stages were isolated in parallel and diluted to the same concentration (4ug/ul). The reference sample was prepared by mixing of the same amount of total RNA from each stage. 25ug of sample and reference RNAs were labeled with either Cy-3 or Cy-5 dUTPs (Amersham, #PA53022 and #PA55022) using SuperScript Direct cDNA Labeling System (Invitrogen, # L1015-02) according to provided protocol. Labeled cDNAs were purified using QIAquick PCR purification kit (QIAGEN, #28104) and combined in one tube. 20ug of sonicated salmon sperm DNA were added. DNA mixture was dried in the stream of nitrogen and dissolved in 60ul of hybridization buffer (50% deionized formamide, 6xSSC, 0.5% SDS and 5xDenhardt’s). Microarray slides were prehybridized in 6xSSC, 0.5% SDS and 1% BSA at 42°C water bath for 1 hour, rinsed with deionized water and dried in bucket rotor centrifuge in 50ml Falcon tubes (5 min, 1000xg). Labeled cDNAs were denatured at 100°C heat block for 2 min, cooled at room temperature, pipetted on the surface of microarray and covered with 25x60 mm LifterSlip (Erie Scientific Company). Assembled microarray was hybridized for 12-14 hours in the VersArray hybridization chamber (Bio-Rad) at 42°C water bath. After hybridization arrays were placed to the beaker with 1xSSC and 0.1% SDS to remove the LifterSlips. Then three washes were performed in the same solution (10 min, 5 min and 5 min) After that microarrays were rinsed with 1xSSC (30 sec), 0.1xSSC (30 sec) and dried in centrifuge as described above. Dry microarrays were scanned at 10 um resolution on ScanArray Lite instrument (Perkin Elmer). The scans were analyzed using TIGR Spotfinder program (www.tm4.org). Data were lowess normalized and filtered using MIDAS software (www.tm4.org). The final datasets were compiled in Microsoft Excel.

Project description:Resurgence of pertussis has been observed in many countries with high vaccination coverage and clonal expansion of certain Bordetella pertussis strains has been associated with recent epidemics in Europe. It is known that vaccinations have selected strains which are different from those used for vaccine production. However, little is known about the differences in genomic content of strains circulating before the vaccination was introduced. In this study, we compared the genomes of 39 vaccine strains and old clinical isolates collected from Finland (N=5), Poland (N=14), Serbia (N=10) and the UK (N=10). The analysis included genotyping, pulsed-field gel electrophoresis (PFGE) and comparative genomic hybridisation (CGH). Compared to the strain Tohama I, the European strains analyzed have lost three major regions of difference (RD3, 5 and 29). However, difference in frequency of the absent RDs 3, 5 or 29 was observed among the four countries. Of the strains with absent RD5, half had also a duplicated region in the genome. All four RDs (RD22, 23, 24 and 26) absent in Tohama I were present in majority of the tested strains. Results obtained from PFGE analysis correlated well with those of CGH. Recently a novel pertussis toxin promoter allele (ptxP3) was described. Strains with ptxP3 have replaced resident ptxP1 strains. When the recent strains (N=22) selected from the four countries were examined, the ptxP3 allele was found in all countries except Poland. Our result indicates that at least three clusters of B. pertussis circulated in Europe in pre-vaccine era and their genomes were distinct from that of the reference strain Tohama I. Although progressive gene loss occurs in B. pertussis population with time, difference in frequency of the lost genes were observed among the countries. The observed differences in genomic content might be vaccine-driven. The strains were serotyped for the fimbriae, genotyped with LightCycler RT-PCR for pertussis toxin subunit 1 (ptxA), pertussis toxin promoter (ptxP) (Kallonen et al 2011, unpublished data, submitted) and pertactin (prn). The vaccine and old strains were assigned to profiles according to the PFGE results. From every PFGE profile from all countries at least 1 strain was selected and tested for CGH. Altogether 29 strains were analysed with CGH from Poland, Serbia and the United Kingdom, 12, 7 and 10, respectively. In addition 5 old previously published Finnish strains were included in the analysis as well as the reference strain Tohama I (Heikkinen et al., 2007). The hybridisations were performed as previously described (Heikkinen et al., 2007). Microarray data analysis was performed with R/Bioconductor (R Development Core Team, 2008). Print-tip-loess normalization was performed with default parameters and the gains/losses were filtered with Limma package. The regions of difference (RDs) found in this study are presented with the nomenclature defined by Brinig et al. (2006). The RDs which were not described by Brinig et al are indicated by RDs and the consequent unused number.

Project description:Prion infection results in progressive neurodegeneration of the central nervous system invariably resulting in death. The pathological effects of prion diseases in the brain are morphologically well defined, such as gliosis, vacuolation, and the accumulation of disease-specific protease-resistant prion protein (PrPSc). However, the underlying molecular events that lead to the death of neurons are poorly characterised. In this study cDNA microarrays were used to profile gene expression changes in the brains of two different strains of mice infected with three strains of mouse-adapted scrapie. Extensive data was collected and analyzed, from which we identified a core group of 349 prion-related genes (PRGs) that consistently showed altered expression in mouse models. Gene ontology analysis assigned many of the up-regulated genes to functional groups associated with one of the primary neuropathological features of prion diseases, astrocytosis and gliosis; protein synthesis, inflammation, cell proliferation and lipid metabolism. Using a computational tool, Ingenuity Pathway Analysis (IPA), we were able to build networks of interacting genes from the PRG list. The regulatory cytokine TGFB1, involved in modulating the inflammatory response, was identified as the outstanding interaction partner for many of the PRGs. The majority of genes expressed in neurons were down-regulated; a number of these were involved in regulatory pathways including synapse function, calcium signalling, long-term potentiation and ERK/MAPK signalling. Two down-regulated genes coding for the transcription regulators, EGR1 and CREB1, were also identified as central to interacting networks of genes; these factors are often used as markers of neuronal activity and their deregulation could be key to loss of neuronal function. These data provides a comprehensive list of genes that are consistently differentially expressed in multiple scrapie infected mouse models. Building networks of interactions between these genes provides a means to understand the complex interplay in the brain during neurodegeneration. Resolving the key regulatory and signaling events that underlie prion pathogenesis will provide targets for the design of novel therapies and the elucidation of biomarkers. Keywords: disease state analysis C57BL/6 mice were inoculated by intracerebral infection of brain homogenate from mice clinically infected with the ME7, 79a and 22A strains of scrapie. In addition VM mice were also inoculated with the 22A scrapie strain. Mice were sacrificed at the onset of clinical diseases as manifested by uncoordinated gait, flaccid paralysis of the hind limbs, rigidity and abolishment of the righting reflex. Brain tissue was collected from these mice and the RNA isolated. Mouse CNS gene expression was analysed by two-colour microarray experiments using an in house manufactured 11K mouse cDNA microarray. RNA from individual infected mice was hybridized to each array versus pooled reference RNA from an equivalent number of age-matched, mock-infected control mice. In total we hybridized 34 different samples to microarrays in this experiment; 8-10 individual mice from each of the four sample groups were individually processed for separate microarrays. Hierachical clustering shows that the patterns of gene expression are for the most part common to the different mouse models . We used the program EDGE to identify genes that were differentially expressed in mouse brain during clinical disease. We used a P value cut-off of 0.05 as the criteria for selection of significantly differentially expressed genes. A similar design was used to determine gene expression profiles from C57BL/6 mice infected with a fourth strain of scrapie, RML. Total RNA from three mice, plus dye swaps, was hybridized to Agilent whole genome mouse arrays to validate genes identified using our manufactured BMAP arrays.

Project description:Changes of genome-wide mRNA transcription levels of human ciliary smooth muscle (hCSM) cells were determined by treating hCSM cells in culture with 200 nM of either an prostaglandin E2 receptor subtype EP2 or subtype EP4 selective agonist for 6 hours in comparison to untreated controls. This was followed by competitive hybridization of fluorescent Cy3 or Cy5 labeled cRNA probes derived from the treated versus untreated control total RNA samples onto an Agilent Human Whole Genome Expression oligonucleotide microarray. Log 2 (LN) of the intra-slide ratios (RATIO, PRE_VALUE) of treated versus untreated samples was reported as VALUE in the sample files. Experiment Overall Design: Human Ciliary Smooth Muscle Cell Culture: Experiment Overall Design: Human ciliary smooth muscle (hCSM) cells were isolated from a female donor, received from the San Diego Eye BankÂ® (San Diego, CA) and cultured in DMEM with 10% fetal bovine serum and 0.5% penicillin/streptomycin according to the method reported previously by Woldemussie et al. Experiment Overall Design: For transcriptome analysis, cells were used for experiments starting at passage 4. Three independent experiments at consecutive cell passages were performed. For each experiment 0.6 x 106 cells were plated onto 15 cm dishes in MEM D-Valine medium (PromoCell, Germany) supplemented with 10% FCS, L-glutamine and antibiotics/antimycotics. Cells were grown to ~80% confluency for 6 days, followed by starvation in absence of FCS and presence of 100 nM of the cyclooxygenase inhibitor indomethacin, 20 Âµg/ml fatty acid-free BSA and 4 Âµg/ml transferrin for 17 hours. Cells were then exposed for 6 hours to 200 nM of either the EP2 agonist AGN210937 or the EP4 agonist AGN202280 (Allergan, Irvine, CA) or the corresponding amount of DMSO in the absence of FCS and presence of transferrin. Experiment Overall Design: Total RNA Extraction: Experiment Overall Design: Total RNA was isolated using a RNA isolation kit (RNAqueous; Ambion, USA) which was followed by DNase I treatment (Turbo DNA-free; Ambion) and purification, according to the manufacturerâ??s protocols. RNA was quantitated using a spectrophotometer ND-1000 (Nanodrop Technologies, USA). RNA quality was assessed regarding purity and stability using a Bioanalyser 2100 (Agilent Technologies, USA). Extracted total RNA aliquots were snap-frozen in liquid nitrogen and stored at -800C for single use. Experiment Overall Design: cRNA Labelling and Oligonucleotide Microarray Hybridization: Experiment Overall Design: Total RNA was linearly amplified and labelled with Cy3 or Cy5 using a low RNA input Fluor Linear Amp Kit (Agilent Technologies, USA). Internal RNA controls from Agilentâ??s RNA Spike-in Kit were included, comprised of mixtures of 11 in vitro synthesized transcripts derived from the Adenovirus E1A transcriptome at various ratios. The final cRNA concentration of 500 ng total RNA used was typically 590-650 ng/Î¼l and the Cy3- or Cy5-cytidine incorporation was 5.4 -6.0 pmol/Î¼g cRNA as determined using the Nanodrop spectrophotometer ND-1000. Experiment Overall Design: 3.5 Î¼g of Cy3 and Cy5 labeled cRNA (treated vs. untreated samples) were competitively hybridized to high-density DNA arrays from Agilent Technologies (Whole Human Genome Oligo Microarray 44K) for 17 hours at 650C according to the manufacturerâ??s protocol. Each set of three microarray experiments for triplicate analysis contained one dye swap experiment. All microarrays were scanned and the intensities normalized over background as well as to eliminate signal intensity-dependent bias from the ratio of the two channels (Lowess normalization) using a microarray scanner from Agilent Technologies including proprietary software. All microarray data sets were imported into the microarray data analysis software Genespring 7.3 (Agilent Technologies) followed by comparison of normalized intensities. Gene identities were updated according to Agilentâ??s latest list of gene annotations (G4112F, 07Feb07). Experiment Overall Design: Consistency of microarray data quality was assessed by monitoring performance of the spiked internal RNA controls (330 microarray spots/array) at various ratios (Supplementary Data, Fig. S1), by carrying out a global analysis of all absolute fluorescent intensity values, and by analysis of negative control spots (314 microarray spots/array) for determination of detection thresholds.

Project description:Exposure to particulate matter (PM) is consistently associated with increased morbidity and mortality attributable in part to respiratory illnesses. The alveolar macrophage (AM) is one of the cell types in the lung constantly exposed to and activated by ambient pollutants. Upon contact with environmental particulate pollutants, AM produces reactive oxygen species (ROS) and antioxidant enzymes, but the scope of this oxidative stress response induced by PM remains poorly defined. In this study, we used microarray analysis to determine the gene expression profile in human alveolar macrophages upon exposure to PM and sought to gain more insight into the global response of pro- and anti-oxidant enzymes to PM exposure. Human AM were obtained by bronchoscopy from normal individuals. They were then incubated with Chapel Hill PM2.5 (1 ïg/ml) or vehicle for 4 hours (n = 6 independent samples each). mRNAs were extracted, amplified and hybridized to Agilent Human 1A microarray. Differentially expressed genes were identified by Statistical Analysis for Microarrays (SAM) with a FDR of 10% and a P â¤ 0.05. Significant genes were also mapped with Gene Ontology (GO) based on their molecular function. We found 34 and 41 up- and down-regulated genes respectively. Of these, 22 genes (~30%) were involved in metal binding and 14 genes were linked to oxidative stress, including 5 metallothionein-1 (MT1) isoform genes. In lung cells, addition of MT-1F in the medium attenuated PM2.5-induced H2O2 production while knockdown of MT1F gene expression increased H2O2 and IL-6 release induced by PM2.5. Our microarray experiments provided a global view of gene expression after in vitro PM2.5 exposure in human AM. The expression profile was most notable for differential expression of genes related to metal binding and oxidative stress, especially upregulation of MT1 isoform genes. Our findings suggest that metals associated with PM, e.g., zinc, copper, and arsenic, induce MT-1 and may be the primary mediators for PM-induced oxidative stress. Keywords: in vitro exposure, PM2.5 particle treatment, human alveolar macrophages Total RNA was isolated from alveolar macrophages from 6 subjects were treated in vitro with PM2.5 particles or vehicle control for 4 hrs. All particle-treated samples were co-hybridized with the common reference sample (pool of all 6 vehicle-treated samples) twice with Cy3 and Cy5 dyes flipped (12 hybridizations). Three samples were also co-hybridized with its own individual control (vehicle-treatment for the same subject) with Cy3 and Cy5 dyes flipped (6 additional hybridizations).

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Transcription profiling of human wildtype DPC4 or empty vector expressed in pancreatic cancer BxPC-3 cell lines

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