Transcription profiling of mouse peritoneal macrophages to test effects of ethanol on poly IC gene induction
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Stephen Pruett
PROVIDER: E-GEOD-1308 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Suppression of innate immunity by acute ethanol administration: a global perspective and a new mechanism beginning with inhibition of signaling through TLR3.
Pruett Stephen B SB Schwab Carlton C Zheng Qiang Q Fan Ruping R
Journal of immunology (Baltimore, Md. : 1950) 20040801 4
Excessive consumption of ethanol (EtOH) suppresses innate immunity, but the mechanisms have not been fully delineated. The present study was conducted to determine whether EtOH suppresses TLR signaling in vivo in mice and to characterize the downstream effects of such suppression. Degradation of IL-1R-associated kinase 1 induced by a TLR3 ligand in peritoneal cells ( approximately 90% macrophages) was suppressed by EtOH. Phosphorylation of p38 kinase in peritoneal macrophages (F4/80(+)) was supp ...[more]
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Project description:Experiment on the effects of ethanol (acute oral) on induction of genes by polyinosinic polycytidylic acid (poly IC). Microarray methodology follows: cDNA for each sample was synthesized from 8.5 µg total RNA using a Superscript cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) in combination with a T7-(dT)24 primer. Following phenol-chloroform extraction of the cDNA, biotinylated cRNA was transcribed in vitro using the BioArray HighYield RNA Transcript Labeling Kit (ENZO Biochem, New York, NY) and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA). Twenty micrograms of purified cRNA was fragmented by incubation in fragmentation buffer (200 mM Tris-acetate, pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate) at 94°C for 35 minutes and chilled on ice. Fifteen micrograms of fragmented biotin-labeled cRNA was hybridized to the Murine Genome U74Av2 Array (Affymetrix, Santa Clara, CA), interrogating a total of 12,488 murine gene cDNA probes, for 16 hr at 45°C with constant rotation (60 rpm). The arrays were washed and then stained for 10 min at 25°C with 10 µg/mL streptavidin-R phycoerythrin (Vector Laboratories, Burlingame, CA) followed by 3 µg/mL biotinylated goat anti-streptavidin antibody (Vector Laboratories) for 10 minutes at 25°C. Arrays were then stained once again with streptavidin-R phycoerythrin for 10 min at 25°C. After washing and staining, the arrays were scanned using an Agilent GeneArray Scanner (Agilent Technologies) at 570 nm. Pixel intensities were measured, expression signals were analyzed and features extracted using a commercial software package (Microarray Suite ver 5.0, Affymetrix). Data mining and statistical analyses were performed with Data Mining Tool ver 3.0 (Affymetrix) algorithms. Arrays were globally scaled to a target intensity value of 2,500 in order to compare individual experiments. The absolute call (present, marginal, absent) of 12,488 gene expressions in each sample, as well as the direction of change, and fold change of gene expressions between samples were identified using the above-mentioned software. Keywords: other
2004-04-15 | GSE1308 | GEO
Project description:Endothelial cells comprise a key component of the inflammatory response. HUVEC (human umbilical vein endothelial cells) were stimulated with interleukin-1 for 0, 0.5, 1, 2.5 and 6 hours, and analyzed using Affymetrix U133A microarrays, in order to obtain a comprehensive overview of the immediate early to early gene expression profiles. HUVEC were isolated and cultured on gelatine-coated cell culture dishes in M199 medium supplemented with 20% FCS, antibiotics, ECGS (endothelial cell growth supplement) and Heparin as described by Zhang et al., Blood 1996;88:3880-3886. HUVEC were stimulated with 100 U/ml human IL-1 (Biosource) for various periods of time, and total RNA isolated using the RNeasy kit (Qiagen) according to the manufacturer's instructions. Hybridization to one set of human U133A GeneChips (Affymetrix, Santa Clara, CA) and scanning of the arrays was carried out according to manufacturerâs protocols (https://www.affymetrix.com). Briefly, 5 µg of total RNA was used to generate double-stranded cDNA by reverse transcription using a cDNA synthesis kit (Superscript Choice System; Life Technologies, Inc., Rockville, MD) that uses an oligo(dT)24 primer containing a T7 RNA polymerase promoter 3' to the polyT (Geneset, La Jolla, CA), followed by second-strand synthesis. Labeled cRNA was prepared from the double-stranded cDNA by in vitro transcription using T7 RNA polymerase in the presence of biotin-11-CTP and biotin-16-UTP (Enzo, Farmington, NY). The labeled cRNA was purified over RNeasy columns (Qiagen, Valencia, CA). Fifteen µg of cRNA was fragmented at 94°C for 35 minutes in 40 mmol/L of Tris-acetate, pH 8.1, 100 mmol/L of potassium acetate, and 30 mmol/L of magnesium acetate. The cRNA was then used to prepare 300 µl of hybridization cocktail (100 mmol/L MES, 1 mol/L NaCl, 20 mmol/L ethylenediaminetetraacetic acid, 0.01% Tween 20) containing 0.1 mg/ml of herring sperm DNA (Promega, Madison, WI) and 500 µg/ml of acetylated bovine serum albumin (Life Technologies, Inc.). Before hybridization, the cocktails were heated to 94°C for 5 minutes, equilibrated at 45°C for 5 minutes, and then clarified by centrifugation (16,000 x g) at room temperature for 5 minutes. Aliquots of this hybridization cocktail containing 15 µg of fragmented cRNA were hybridized to HU133A arrays (Affymetrix, Santa Clara, CA) at 45°C for 16 hours in a rotisserie oven at 60 rpm. The arrays were washed using non-stringent buffer (6xSSPE: 0.9 M sodium chloride, 0.06 M sodium phosphate, 6 mM EDTA pH 7.4) at 25°C, followed by stringent buffer (100 mmol/L MES, pH 6.7, 0.1 mol/L NaCl, 0.01% Tween 20) at 50°C. The arrays were stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR), washed with 6xSSPE, incubated with biotinylated anti-streptavidin IgG, stained again with streptavidin-phycoerythrin, and washed again with 6xSSPE. The arrays were scanned using the GeneArray scanner (Affymetrix). Image analysis was performed with GeneChip software (Affymetrix, MAS 5.0). Normalization was performed by global scaling, with the arrays scaled to an average intensity of 500.
2008-06-16 | E-GEOD-973 | biostudies-arrayexpress
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2004-07-20 | GSE973 | GEO
Project description:Rationale: During excessive pressure or overload, cardiac cells are subjected to increased mechanical stress. We investigate how the stress response of cardiac cells to mechanical stress can be compared to genotoxic stresses induced by DNA damaging agents. Methods: Cultures enriched for cardiomyocytes and cultures of fibroblasts were derived from ventricles of neonatal rat hearts. In three independent experiments, both cell cultures were subjected to mechanical stress (cyclic stretch at 60 cycles/min), UV irradiation (10 J/m2), or X-rays (8.5 Gy). Cells were harvested 24 h. after (the onset of) treatment. Total RNA was isolated using an RNeasy® kit (Qiagen). Single stranded cDNA was synthesized from RNA from myocyte-enriched populations (10-14 microgram) and populations of fibroblasts (7.5-14 microgram), using Superscript II reverse transcriptase and T7-oligo(dT)24 primers at 42ºC for 1 h. Double stranded cDNA was obtained by using DNA ligase, DNA polymerase I and RNAse H at 16ºC for 2 h, followed by T4 DNA polymerase at 16ºC for 5 min. cRNA was synthesized from cDNA using the BioArray HighYield® RNA transcript labeling kit (Enzo) in the presence of biotin-labeled CTP and UTP. cRNA was fragmented in a buffer containing 40 mM Tris-acetate (pH 8.1), 100 mM KAc and 30 mM MgAc, at 94ºC for 35 min. Fragmented cRNA was hybridized to GeneChip RG-U34A arrays (Affymetrix) at 45ºC for 16 h. Arrays were washed and incubated with a streptavidin-phycoerythrin complex. Fluorescent signals were determined with a GeneChip scanner. Keywords: other
2004-12-03 | GSE2032 | GEO
Project description:Peripheral blood mononuclear cells were isolated 0, 30 min, 6 h, 24 h, and 7 day after intravenous endotoxin challenge using cell preparation tubes (Vacutainer® CPT). The PBMC layer was lysed with RLT buffer, homogenized, and then stored at 80°C. Total RNA was extracted as per RNeasy Midi protocol (Qiagen). Double stranded cDNA was synthesized from total RNA (5 to 20 µg) using 7-d(T)24 primer and SuperScript Double-Stranded cDNA Synthesis Kit and purified by phase lock gel-phenol/chloroform extraction followed by ethanol precipitation. Biotin-labeled cRNA was prepared by in vitro transcription using HighYield RNA Transcript Labeling Kit followed by fragmentation with 5X fragmentation buffer. Fragmented cRNA (10 µg) was hybridized to Affymetrix Hu95Av2 oligonucleotide probe arrays for 16 h at 45?C. After removal of hybridization fluid, the arrays were washed, stained with streptavidin phycoerythrin (SAPE), and signal amplified by anti-streptavidin antibody using Affymetrix Fluidics Station 400. Probe set signals were measured using Agilent GeneArray Scanner (Affymetrix, Santa Clara, CA).
2007-12-19 | E-GEOD-3026 | biostudies-arrayexpress
Project description:TGF-beta1 is a pleiotropic cytokine that regulates multiple cellular functions including cell proliferation, differentiation, and apoptosis. It has been identified as one of the mediators of renal hypertrophy and accumulation of extracellular matrix proteins in the diabetic glomerular mesangium. Previous results from our and other laboratories indicate that TGF-beta1 mediates some of the effects of high glucose and glucosamine on extracellular matrix gene expression in mesangial cells. The global regulatory mechanism(s), however, still remains to be understood. In this study, we have utilized an in vitro model of mouse mesangial cells (MES-13) to investigate the genetic effects of TGF-beta1 on global patterns of gene expression, transcriptome and signaling networks. A total number of 179 genes are regulated by TGF-beta1 in MES-13 cells at 2.0-fold, which cover across 44 physiological processes, 31 metabolic functions, and 11 major signaling pathways. We observed that several genes regulated by high glucose and glucosamine are also targeted by TGF-beta1 in MES-13 cells such as thioredoxin interacting protein, glutathione S-transferase theta 1, selenium binding protein 1, inhibitor of DNA binding 2, and plasminogen activator inhibitor-1. The results of this study further support the hypothesis that some effects of high glucose and glucosamine on mesangial gene expression are mediated by TGF-beta1. The identification of these common genes will help us to investigate further their roles in the development and progression of diabetic kidney disease. Cell culture: Stable murine mesangial (MES-13) cells transformed with non-capsid-forming SV-40 virus were obtained from the ATCC, Manassas, VA. These cells display a differentiated mesangial cell phenotype including the typical spindle-like appearance, positive staining for vimentin and desmin, and contraction in response to ANG II and expression of AT1 receptor. The cells were maintained in DMEM and F-12 Nutrient Mixture (Ham's) (4:1 ratio) (GIBCO BRL, Gaithersburg, MD) containing a normal D-glucose concentration of 5.5 mmol/L, 2% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin, and 2 mmol/L glutamine (26). The cells were incubated in a humidified incubator of 5% CO2 at 37 °C and routinely passaged at confluence every 3 days by trypsinization using 10-cm culture dishes. Approximately 50% confluent monolayers were starved in the above medium without FCS for 1 day and then incubated in the starvation medium with 100 ng/ml TGF-beta1 for 24hrs. Total RNA isolation, cDNA and cRNA synthesis and genechip hybridization: Total RNA was isolated using Trizol reagent (Life Technologies, Inc., Massachusetts). cDNA and cRNA synthesis, genechip hybridization, and scanning of the Affymetrix murine expression U430 2.0 chips (the Affymetrix M430 2.0 chip contains 39,000 transcripts targeted at 34,000 well characterized genes) were performed according to the manufacturer's protocol (Affymetrix, Santa Clara, CA). Briefly, 5 mg of RNA was converted into double-stranded cDNA by reverse transcription using a cDNA synthesis kit (SuperScript Choice, Life Technologies, Inc., MA) with an oligo(dT)24 primer containing a T7 RNA polymerase promoter site added 3' of the poly(T) (Genset, La Jolla, CA). After second-strand synthesis, labeled cRNA was generated from the cDNA sample by an in vitro transcription reaction supplemented with biotin-11-CTP and biotin-16-UTP (Enzo, Farmingdale, NY). The labeled cRNA was purified by using RNeasy spin columns (Qiagen, Valencia, CA). 15 mg of each cRNA was fragmented at 94 °C for 35 min in fragmentation buffer (40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate) and then used to prepare 300 ml of hybridization mixture (100 mM MES, 0.1 mg/ml herring sperm DNA (Promega), 1M sodium chloride, 10 mM Tris, pH 7.6, 0.005% Triton X-100) containing a mixture of control cRNAs samples were heated at 94 °C for 5 min, equilibrated at 45 °C for 5 min, and clarified by centrifugation (14,000 x g) at room temperature for 5 min. Aliquots of each sample (10 mg of cRNA in 200 ml of the master mix) were hybridized to GeneChip Mouse Genome 430 2.0 Array at 45 °C for 16 h in a rotisserie oven set at 60 rpm, then washed with non stringent wash buffer (6x saline/sodium phosphate/EDTA) at 25 °C, followed by stringent wash buffer (100 mM MES (pH 6.7), 0.1 M NaCl, 0.01% Tween 20) at 50 °C, stained with streptavidin-phycoerythrin (Molecular Probe), washed again with 6x saline/sodium phosphate/EDTA, stained with biotinylated anti-streptavidin lgG, followed by a second staining with streptavidin-phycoerythrin, and a third washing with 6x saline/sodium phosphate/EDTA using the GeneChip Fluidics Station 450. The arrays were then scanned using a GeneChip Scanner 3000 (Affymetrix). Each experiment was repeated twice. Microarray Data analysis: The chips were read with Affymetrix GCOS v1.2, and the probe intensity files were modeled with DChip 1.3 (Harvard School of Public Health) in both PM-only and PM-MM modes. Consensus differentially regulated genes were initially derived from repeat experiments on the bases of a 90% CI of greater than 2-fold change in expression and with p<0.05 of error in paired t-test across repeats. This was further validated through modeling of variance between sample groups (one-way ANOVA) conducted in GeneSpring 6.0 (Silicon Genetix). The consensus gene-set was clustered in DChip and GeneSpring 6.0, with OntoExpress (Wayne State University) to explore ontological associations. Further ontological and pathway analyses were conducted with the GeneGo software and NIH's DAVID bioinformatics programs (heep://appls.niaid.nih.gov/david). Keywords = TGFBeta1 Keywords = mesangial cells Keywords = diabetic glomerulopathy Keywords: repeat sample
2006-01-01 | GSE2558 | GEO
Project description:High quality RNA was extracted from the whole seedlings (Combined root and leaf samples) using TRI Reagent (Ambion, Inc. USA) and pooled from 12 independent stressed and non-stressed plant samples separately, and treated with DNase-I (QIAGEN GmbH, Germany). Subsequently, RNA cleanup was carried out using RNeasy Plant Mini Kit (QIAGEN GmbH, Germany) and 5 ug of total RNA from each sample in triplicates were reverse-transcribed to double stranded cDNA using the GeneChipï¾® One-Cycle cDNA Synthesis Kit. The biotin-labelled cRNA was made using the GeneChipï¾® IVT Labelling Kit (Affymetrix, CA, USA). Twenty microgram of cRNA samples was fragmented and out of which which 7.5 ug cRNA were hybridized for 16 hours at 45C to the Affymetrix GeneChipï¾® Rice Genome Array (Santa Clara, CA, USA). After washing and staining with R-phycoerythrin streptavidin in a Fluidics Station, using the Genechipï¾® Fluidics Station 450, the arrays were scanned by the Genechipï¾® 3000 Scanner. The chip images were scanned and extracted using default settings and the CEL files were produced with the Affymetrix GeneChip Operating Software (GCOS 1.2). The resulting .CEL files were imported into the GeneSpring GX 10 (Agilent Technologies Inc, Santa Clara CA) and normalized with the PLIER16 algorithm. The resulting expression values were log2-transformed. Average log signal intensity values of three technical replicates for each sample were used for advance analysis.
2010-10-09 | E-MEXP-2401 | biostudies-arrayexpress
Project description:A total of 20 KYSE human esophageal squamous cell carcinoma cell lines (KYSE-30, -140, -150, -170, -180, -200, -220, -350, -410, -450, -510, -520, -590, -770, -850, -890, -1170, -1190, -1250, and -2270) were kindly provided by Dr. Y. Shimada (Kyoto University, Kyoto, Japan). The KYSE cell lines were cultured in RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (FBS; BioWhittaker, Verviers, Belgium) in a humidified atmosphere of 5% CO2 and maintained in continuous exponential growth by passage every 3 days. The exponentially growing cultured cells (2 x 10^6) were collected after two-washings with PBS. Total RNA was prepared from the frozen cell pellets using QIAGEN RNeasy mini kit (QIAGEN, Inc., Valencia, CA). The quality of the RNA was checked using Agilent Technologies 2100 Bioanalyzer (Agilent, Palo Alto, CA). CodeLink Expression Bioarray System (Amersham Bioscience, Tokyo, Japan) was used according to the manufacturer’s protocol. Briefly, first-strand cDNA was generated from 1 µg of total RNA for each cell line using reverse transcriptase and a T7 primer, and then second-strand cDNA was produced using DNA polymerase mix and RNase H. cRNA was generated via an in vitro transcription reacrion using T7 RNA polymerase and biotin-11-UTP (Perkin Elmer, Boston, MA). After quantified by spectrometry and qualified using Agilent Technologies 2100 Bioanalyzer (Agilent), 10 µg of cRNA was fragmented and hybridized to a Uniset Human 20K I Bioarray containing 19,981 human probes with 108 positive and 300 negative bacterial control probes. After hybridization, the arrays were rinsed and labeled with Streptavidin-Cy5, scanned using Agilent DNA Microarray Scanner (Agilent), then analyzed with CodeLink Expression Analysis Software ver.2.3. Expression levels were normalized to the median expression value of the whole array spots except for the bacterial controls. Keywords: parallel sample
2005-12-31 | GSE2447 | GEO
Project description:TGF-beta1 is a pleiotropic cytokine that regulates multiple cellular functions including cell proliferation, differentiation, and apoptosis. It has been identified as one of the mediators of renal hypertrophy and accumulation of extracellular matrix proteins in the diabetic glomerular mesangium. Previous results from our and other laboratories indicate that TGF-beta1 mediates some of the effects of high glucose and glucosamine on extracellular matrix gene expression in mesangial cells. The global regulatory mechanism(s), however, still remains to be understood. In this study, we have utilized an in vitro model of mouse mesangial cells (MES-13) to investigate the genetic effects of TGF-beta1 on global patterns of gene expression, transcriptome and signaling networks. A total number of 179 genes are regulated by TGF-beta1 in MES-13 cells at 2.0-fold, which cover across 44 physiological processes, 31 metabolic functions, and 11 major signaling pathways. We observed that several genes regulated by high glucose and glucosamine are also targeted by TGF-beta1 in MES-13 cells such as thioredoxin interacting protein, glutathione S-transferase theta 1, selenium binding protein 1, inhibitor of DNA binding 2, and plasminogen activator inhibitor-1. The results of this study further support the hypothesis that some effects of high glucose and glucosamine on mesangial gene expression are mediated by TGF-beta1. The identification of these common genes will help us to investigate further their roles in the development and progression of diabetic kidney disease. Cell culture: Stable murine mesangial (MES-13) cells transformed with non-capsid-forming SV-40 virus were obtained from the ATCC, Manassas, VA. These cells display a differentiated mesangial cell phenotype including the typical spindle-like appearance, positive staining for vimentin and desmin, and contraction in response to ANG II and expression of AT1 receptor. The cells were maintained in DMEM and F-12 Nutrient Mixture (Ham's) (4:1 ratio) (GIBCO BRL, Gaithersburg, MD) containing a normal D-glucose concentration of 5.5 mmol/L, 2% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin, and 2 mmol/L glutamine (26). The cells were incubated in a humidified incubator of 5% CO2 at 37 °C and routinely passaged at confluence every 3 days by trypsinization using 10-cm culture dishes. Approximately 50% confluent monolayers were starved in the above medium without FCS for 1 day and then incubated in the starvation medium with 100 ng/ml TGF-beta1 for 24hrs. Total RNA isolation, cDNA and cRNA synthesis and genechip hybridization: Total RNA was isolated using Trizol reagent (Life Technologies, Inc., Massachusetts). cDNA and cRNA synthesis, genechip hybridization, and scanning of the Affymetrix murine expression U430 2.0 chips (the Affymetrix M430 2.0 chip contains 39,000 transcripts targeted at 34,000 well characterized genes) were performed according to the manufacturer's protocol (Affymetrix, Santa Clara, CA). Briefly, 5 mg of RNA was converted into double-stranded cDNA by reverse transcription using a cDNA synthesis kit (SuperScript Choice, Life Technologies, Inc., MA) with an oligo(dT)24 primer containing a T7 RNA polymerase promoter site added 3' of the poly(T) (Genset, La Jolla, CA). After second-strand synthesis, labeled cRNA was generated from the cDNA sample by an in vitro transcription reaction supplemented with biotin-11-CTP and biotin-16-UTP (Enzo, Farmingdale, NY). The labeled cRNA was purified by using RNeasy spin columns (Qiagen, Valencia, CA). 15 mg of each cRNA was fragmented at 94 °C for 35 min in fragmentation buffer (40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate) and then used to prepare 300 ml of hybridization mixture (100 mM MES, 0.1 mg/ml herring sperm DNA (Promega), 1M sodium chloride, 10 mM Tris, pH 7.6, 0.005% Triton X-100) containing a mixture of control cRNAs samples were heated at 94 °C for 5 min, equilibrated at 45 °C for 5 min, and clarified by centrifugation (14,000 x g) at room temperature for 5 min. Aliquots of each sample (10 mg of cRNA in 200 ml of the master mix) were hybridized to GeneChip Mouse Genome 430 2.0 Array at 45 °C for 16 h in a rotisserie oven set at 60 rpm, then washed with non stringent wash buffer (6x saline/sodium phosphate/EDTA) at 25 °C, followed by stringent wash buffer (100 mM MES (pH 6.7), 0.1 M NaCl, 0.01% Tween 20) at 50 °C, stained with streptavidin-phycoerythrin (Molecular Probe), washed again with 6x saline/sodium phosphate/EDTA, stained with biotinylated anti-streptavidin lgG, followed by a second staining with streptavidin-phycoerythrin, and a third washing with 6x saline/sodium phosphate/EDTA using the GeneChip Fluidics Station 450. The arrays were then scanned using a GeneChip Scanner 3000 (Affymetrix). Each experiment was repeated twice. Microarray Data analysis: The chips were read with Affymetrix GCOS v1.2, and the probe intensity files were modeled with DChip 1.3 (Harvard School of Public Health) in both PM-only and PM-MM modes. Consensus differentially regulated genes were initially derived from repeat experiments on the bases of a 90% CI of greater than 2-fold change in expression and with p<0.05 of error in paired t-test across repeats. This was further validated through modeling of variance between sample groups (one-way ANOVA) conducted in GeneSpring 6.0 (Silicon Genetix). The consensus gene-set was clustered in DChip and GeneSpring 6.0, with OntoExpress (Wayne State University) to explore ontological associations. Further ontological and pathway analyses were conducted with the GeneGo software and NIH's DAVID bioinformatics programs (heep://appls.niaid.nih.gov/david).
2008-06-12 | E-GEOD-2558 | biostudies-arrayexpress
Project description:We have used commercially available oligonucleotide arrays to measure the relative expression levels of >10,000 genes and ESTs. Recent evidence suggested certain advantages of performing such expression experiments on two or more different platforms of oligonucleotide arrays. Here we have used two oligonucleotide platforms, MG U74Av2.0 arrays from Affymetrix and CodeLink UniSet1 from GE Healthcare, to assess whole brain gene expression patterns in naive C57BL/6 and DBA/2J mice. C57BL/6J (n = 5) and DBA/2J (n = 4) male mice, 25-30 g body weight, were obtained from Jackson Laboratories. Animals were group-housed and quickly sacrificed by CO2 exposure according to a protocol approved by the UCHSC IACUC. Brains were rapidly removed and stored at -80oC until use. Whole brain was homogenized in lysis buffer using a Polytron, and total RNA was isolated using the RNeasy Midi Kit (Qiagen) following the protocol supplied by the manufacturer. An additional clean-up of total RNA was carried out using the RNeasy Mini Kit (Qiagen). Using the protocol supplied by the manufacturer, double-stranded cDNA was synthesized from the total RNA (4 ug of total RNA from an individual mouse brain was used for each array) and was used to obtain biotin-labeled cRNA by an in vitro transcription reaction. Biotin-labeled cRNA was recovered using the RNeasy kit. Biotin-labeled cRNA from each mouse was then fragmented and hybridized with two separate (duplicate) CodeLink BioArrays. The average of the gene expression intensity values for each probe set from these technical replicates was used for data analysis. The BioArrays were subsequently stained for 30 min with Cy5-Streptavidin and washed prior to scanning. For MG U74Av2 arrays,double-stranded cDNA was synthesized from 5 ug of total RNA, using the protocol supplied by the manufacturer, and used to obtain biotin-labeled cRNA by an in vitro transcription reaction. Biotin-labeled cRNA was then fragmented and the quality of the fragmented cRNA was assessed using Test2 chips prior to hybridization with the Affymetrix arrays. Hybridization of the cRNA with the Affymetrix arrays was carried out according to the manufacturer's protocol. The arrays were stained with streptavidin-phycoerythrin conjugate and scanned by a GeneArray scanner. Keywords: other
2006-04-05 | GSE2502 | GEO