Strain differences in copper sulfate
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE1073 | GEO | 2004/03/01
SECONDARY ACCESSION(S): PRJNA87167
REPOSITORIES: GEO
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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.) Keywords: Time course
2006-09-22 | GSE5892 | GEO
Project description:We used oligonucleotide microarrays to find differentially expressed genes between control subjects and those affected by sporadic amyotrophic lateral sclerosis. Experiment Overall Design: Complementary RNAs (cRNAs) labelled with Cy5-CTP (Perkin-Elmer) were synthesized from 1 µg of total RNA of each sample of human motor cortex using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies) following the manufacturerâs protocol. A reference cRNA, labelled with Cy3-CTP (Perkin-Elmer), was synthesized from 1 µg of sample Diseased 2 RNA. Aliquots (750 ng) of Cy3 and Cy5 labelled cRNA targets were co-hybridized on Whole Human Genome Oligo Microarrays (Agilent Technologies). Microarray hybridization and washing were performed using reagents and instruments (hybridization chambers and rotating oven) as indicated by the manufacturer (Agilent Technologies). Microarrays were scanned at 10-μm resolution using a GenePix Personal 4100A microarray scanner and the GenePix Pro 6.0 acquisition and data-extraction software (Molecular Devices, Corp.).
2008-06-13 | E-GEOD-4595 | biostudies-arrayexpress
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Project description:Gene expression of adherent cells in a coculture of the human bone marrow stromal cell line HS-27a and peripheral blood monocytes (CD14+ cells) was compared to HS-27a cultured alone. Microarray experiments were conducted using CD14+ cells from 7 healthy donors over a period of >7 months. Monocytes were isolated by ficoll separation of whole blood, followed by labelling cells with CD14 monoclonal antibody Tuek4 and magnetic bead selection (Milteny rat anti mouse IgG2a+2b beads) on an AutoMacs. HS27a cells were plated in T75 flasks at approximately 80% confluence. The next day 1.5x10E6 freshly isloated human CD14+ cells were added to the culture. At 3 days the cultures were washed 3x with Hank’s buffer to remove nonadherent monocytes. Co-cultures and HS27a cultured alone were harvested by brief trypsinization followed by pelleting of the cells. All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 ug) was annealed with 5 ug oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42ºC in the presence of 0.5mM dGTP, 0.5mM dCTP, 0.5mM dATP, 0.3mM dTTP, 0.2mM amino-allyl dUTP. After hydrolysis of RNA in 0.2M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from HS27a and HS27a-monocyte cocultures was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit and suspended in 36 ul of 3X SSC and 0.8 mg/ml of poly-A for hybridization to the microarray. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction and removal of flagged values, log base 2 expression ratios were mean centered and linear transformed to obtain the log and linear values given in the data tables. Keywords: repeat sample
2003-12-22 | GSE831 | GEO
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2020-10-20 | PXD021205 | Pride
Project description:Himalaya v 292 Four biological replicates were used for the comparison and the dyes were swapped among the test and control samples for labeling two of the replicates. For each replicate, 50 ?g of total RNA was used for both the Cy3 and Cy5 dyes (Amersham Pharmacia, UK), following the two-step labeling method of Schenk et al. (2000). The control plant, 'Himalaya' was labeled with Cy3 in the un-swapped replicates. The dyes were reversed among the replicate experiments to minimize any bias in cDNA incorporation and photo-bleaching of the fluorescent dyes. The pre-hybridization of the microarray slides, hybridization of samples and subsequent washing of the slides to remove unbound target was performed as per the supplied protocol for the CMT-GAPSTM coated microscope slides (Corning USA). The slides were scanned with a GenePix 4000A microarray scanner (Axon Instruments, Union, CA, USA). The features were analyzed using the GenePix Pro 4 software and unsatisfactorily segmented features were either manually adjusted or discarded to ensure the integrity of the data obtained.
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2004-05-16 | GSE1310 | GEO
Project description:a- Self –Self Hybridisation were used to set confidence 99% interval using RNA from non-tethered cell lines that is both labelled in with cy3 cy5 (theoretically identical cDNA populations) to compared technical errors associated with such experiments Keywords: comparative hybridization to access expression profiles between Cy3 and Cy5 uniformally labelled template.
2008-03-04 | GSE10697 | GEO