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Gene expression profile of human lung cell line (A549) treated with 2mM 59cobalt chloride during 30mn, 2h, 4h and 24h

ABSTRACT: The purpose of this work was to identify the potential signatures of cobalt exposure using a toxicogenomic approach. A time course transcriptome analysis was performed on human type II epithelial cell line A549. Cells were exposed to cobalt at midlog phase ; a medium without FCS containing cobalt (CoCl2, Sigma) or not was added for 30mn, 2h, 4h or 24h. Total RNA was isolated using the Quiagen RNeasy miniprep kit, and then labelled using the FairPlay Microarray Labelling Kit (Stratagene). Amino reactive Cy3- and Cy5- dyes (CyTMDye Post-Labelling Reactive Dye Pack, Amersham) were further chemically coupled to test and control cDNA respectively. After hybridization, microarrays were scanned with GenePix 4000B (Axon Instrument Inc., Forster City, CA). Cy3 and Cy5 fluorescence intensities for the spots were quantified after local subtraction of background noise using Genepix Pro 4.0 software (Axon Instrument Inc.) Keywords: Time course

ORGANISM(S): Homo sapiens

PROVIDER: GSE5892 | GEO | 2006/09/22

SECONDARY ACCESSION(S): PRJNA97319

REPOSITORIES: GEO

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Gene expression profile of human lung cell line (A549) treated with 2mM 59cobalt chloride during 30mn, 2h, 4h and 24h

Project description:The purpose of this work was to identify the potential signatures of cobalt exposure using a toxicogenomic approach. A time course transcriptome analysis was performed on human type II epithelial cell line A549. Cells were exposed to cobalt at midlog phase ; a medium without FCS containing cobalt (CoCl2, Sigma) or not was added for 30mn, 2h, 4h or 24h. Total RNA was isolated using the Quiagen RNeasy miniprep kit, and then labelled using the FairPlay Microarray Labelling Kit (Stratagene). Amino reactive Cy3- and Cy5- dyes (CyTMDye Post-Labelling Reactive Dye Pack, Amersham) were further chemically coupled to test and control cDNA respectively. After hybridization, microarrays were scanned with GenePix 4000B (Axon Instrument Inc., Forster City, CA). Cy3 and Cy5 fluorescence intensities for the spots were quantified after local subtraction of background noise using Genepix Pro 4.0 software (Axon Instrument Inc.) 30 mn : exposure 1 (2 microarrays) - exposure 2 (4 microarrays) 2h : exposure 1 (2 microarrays) - exposure 2 (4 microarrays) 4h : exposure 1 (2 microarrays) - exposure 2 (2 microarrays) 24h: exposure 1 (4 microarrays) - exposure 2 (6 microarrays) - exposure 3 (4 microarrays)

2006-09-22 | E-GEOD-5892 | biostudies-arrayexpress

Responses of HUVEC to Hypoxia and Reoxygenation

Project description:Human umbilical vein endothelial cells (HUVEC) were cultivated on 1%-gelatin-coated 75-cm2 culture flasks in Medium 200 supplemented with 2% fetal calf serum, penicillin (100 units/ml), streptomycin (100 units/ml) and Funizone (0.25 μg/ml) supplied by Cascade Biologics (Portland, OR). Prior to an experiment, HUVECs were subcultivated with Trypsin/EDTA onto 1% gelatin coated glass 30-mm glass coverslips in 60 mm glass Petri dishes. The hypoxic treatment was performed in airtight chambers from Mitsubishi Gas Chemical Co. Inc (Remel Scientific, Baton Rouge, LA) Preconditioning consisted of 1-hour hypoxia followed by various periods of reoxygenation (0, 3, 5, 12 and 24 hrs) for gene expression analysis. Total RNA was extracted from cultured HUVECs with TRITM reagent according to the manufacture’s instructions (Molecular Research Center, Cincinnati, OH). Cy5 and Cy3 labeled cDNA probes were generated from total RNA by reverse transcription in the presence of aminoallyl-dUTP according to the method described by Xiang, et al,15. Human universal reference RNA (Stratagene, La Jolla, CA) was Cy3-labeled and used as reference RNA and RNA isolated from HUVEC at 0, 3, 5, 12 and 24 hour following reoxygenation were Cy5-labeled. The high-resolution scans of hybridized microarrays were acquired with a GenePix scanner 4000B (Axon Instruments, Inc., Union City, CA) and tabulated with GenePix pro 5.1 software. Keywords: time-course

2004-02-18 | GSE1041 | GEO

Transcription profiling of background variability of HFFs

Project description:Background variability was determined by hybridizing Cy3- and Cy5-cDNA from the same source (mock infected HFFs) to arrays (HE series). Polyadenylated RNA from 107 confluent HFs was purified by using Oligotex (QIAGEN, Valencia, Calif.) from Trizol (Invitrogen)-extracted total RNA. This RNA was prepared, reverse transcribed, labeled with Cy3-dUTP or Cy5-dUTP (Amersham, Little Chalfont, Buckinghamshire, United Kingdom) by random primed synthesis with DNA pol I Klenow (Amersham Life Science, Inc., Cleveland, Ohio), and hybridized to spotted, human cDNA microarrays as previously described. Sequence-verified human cDNA microarrays (HE and HG series, 31,000 spots; HD51 series, 17,000 spots) were produced at Stanford. Images were collected by using a GenePix 4000B microarray scanner, manually flagged to eliminate poor spots and analyzed by GenePix Pro 2.0 (Axon, Union City, Calif.).

2005-11-07 | E-SMDB-2961 | biostudies-arrayexpress

Transcription profiling of wild type vs pp65 mutant CMV infected HFFs

Project description:Polyadenylated RNA from 107 confluent HFs was purified by using Oligotex (QIAGEN, Valencia, Calif.) from Trizol (Invitrogen)-extracted total RNA. This RNA was prepared, reverse transcribed, labeled with Cy3-dUTP or Cy5-dUTP (Amersham, Little Chalfont, Buckinghamshire, United Kingdom) by random primed synthesis with DNA pol I Klenow (Amersham Life Science, Inc., Cleveland, Ohio), and hybridized to spotted, human cDNA microarrays as previously described. Sequence-verified human cDNA microarrays (HE and HG series, 31,000 spots; HD51 series, 17,000 spots) were produced at Stanford. Images were collected by using a GenePix 4000B microarray scanner, manually flagged to eliminate poor spots and analyzed by GenePix Pro 2.0 (Axon, Union City, Calif.).

2005-11-07 | E-SMDB-2962 | biostudies-arrayexpress

Transcription profiling of human wild type CMV vs mock infected HFFs

Project description:Polyadenylated RNA from 10^7 confluent HFs was purified by using Oligotex (QIAGEN, Valencia, Calif.) from Trizol (Invitrogen)-extracted total RNA. This RNA was prepared, reverse transcribed, labeled with Cy3-dUTP or Cy5-dUTP (Amersham, Little Chalfont, Buckinghamshire, United Kingdom) by random primed synthesis with DNA pol I Klenow (Amersham Life Science, Inc., Cleveland, Ohio), and hybridized to spotted, human cDNA microarrays as previously described. Sequence-verified human cDNA microarrays (HE and HG series, 31,000 spots; HD51 series, 17,000 spots) were produced at Stanford. Images were collected by using a GenePix 4000B microarray scanner, manually flagged to eliminate poor spots and analyzed by GenePix Pro 2.0 (Axon, Union City, Calif.)

2005-11-07 | E-SMDB-2957 | biostudies-arrayexpress

Transcription profiling of human pp65 mutant CMV vs mock infected HFFs

2005-11-07 | E-SMDB-2959 | biostudies-arrayexpress

Transcription profiling of wild type vs pp65 mutant CMV infected HFFs

2005-11-07 | E-SMDB-2960 | biostudies-arrayexpress

Comparison between Saccharomyces cerevisiae HS 10, HS 34 and HS 12, HS 37

Project description:Gene expression of different flocculent (HS 10 and HS 34) and powdery (HS 12 and HS 37) yeast strains compared to each other during exponential and stationary growth phase was analysed. The isolation of RNA was done by disruption of the cells under liquid nitrogen using mortar and pistil and then the Qiagen RNeasy Midi Kit with some modifications within the manufacturers´ protocol and the Qiagen RNase-Free DNase Set were applied. 6 µg of the total RNA per sample was used for each microarray experiments. The indirect labelling by the tyramide-signal-amplification method (MicromaxTM TSATM labelling and detection Kit from Perkin Elmer life sciences) was used to increase the Cy3 and Cy5 signals of microarray detection. Each cDNA containing Biotin- and Fluorescein-nucleotides respectively was purified with a QIAquick PCR purification kit and suspended in 11 µl of the formamide containing hybridization buffer. The slides were hybridized at 42°C over night under a cover slip. The microarrays were scanned by the Axon 4000B scanner; image intensity data were extracted and analysed with GenePix® Pro 6.0 software. Data from different scans of Dye-swap experiment were extracted by GenePix Pro 6.0 software, normalized and united. An outliertest has been applied in order to find outliers amongst the gene replicats. Subsequently, a t-test (1% and 5% probability of error) has been used in order to find regulated genes. Keywords: sorted yeast cells

2008-12-12 | GSE12369 | GEO

Saccharomyces cerevisiae sorted and untreated

Project description:Gene expression of a vital, stained and sorted subpopulation of Saccharomyces cerevisiae with high affinity to glucose, harvested at a dilution rate of D=0.160 h-1, and of cells from the whole population without further treatment grown at the same dilution rate were analysed. The isolation of RNA was accomplished by using lyticase to lyse the cells and the Qiagen RNeasy Mini Kit with some modifications within the manufacturers´ protocol. 6 µg of the total RNA per sample was used for each microarray experiments. The indirect labelling by the tyramide-signal-amplification method (MicromaxTM TSATM labelling and detection Kit from Perkin Elmer life sciences) was used to increase the Cy3 and Cy5 signals of microarray detection. Each cDNA containing Biotin- and Fluorescein-nucleotides respectively was purified with a QIAquick PCR purification kit and suspended in 11 µl of the formamide containing hybridization buffer. The slides were hybridized at 42°C over night under a cover slip. The microarrays were scanned by the Axon 4000B scanner; image intensity data were extracted and analysed with GenePix® Pro 6.0 software. Data from different scans of Dye-swap experiment were extracted by GenePix Pro 6.0 software, normalized and united. An outliertest has been applied in order to find outliers amongst the gene replicats. Subsequently, a t-test (1% and 5% probability of error) has been used in order to find regulated genes. Keywords: sorted yeast cells

2007-10-01 | GSE8247 | GEO

P. aeruginosa PAO wildtype Cy3 PA3898 mutant Cy5

Project description:P. aeruginosa PAO wildtype Cy3 PA3898 mutant Cy5

| PRJNA491282 | ENA