title: 22-oct-2002-BrainDsubA-ab150

description:
Dietary effects of arachidonate-rich fungal oil and fish oil on murine hepatic and hippocampal gene expression, Lipids in Health and Disease 2002.
Diet contained 90% fat-free AIN93G rodent diet in powder form (Lot 9350-5, Dyets, Inc., Bethlehem, PA), 0.4% milk fat, 1.2% palm olein, 1.9% Trisun sunflower oil, 1.5% soybean oil and 2.1-5.1% medium chain triacylglycerol oil (all of the above ingredients except the AIN93G were from Nestlé affiliated companies). Part of the medium chain triacylglycerol oil in control diet was replaced with: 1.1% fungal oil (providing 0.5 dietary wt% AA and 1.0en%). Food was maintained at -80°C in daily aliquots under liquid N2, thawed each afternoon before administration to mice, and uneaten food was discarded daily.
Prior to actual experimentation, test dissections were made on identical mice to perfect the dissection techniques at Porsolt & Partners Pharmacology (Paris, France).
Male Rj:NMRI mice from Elevage Janvier, Le Genest-Saint-Isle France, weighing 10-11 g at delivery and 33-51 grams on experimental d 42, were housed 10 per cage in wire cages with bedding and normal light cycle. Mice received ad libitum quantities of distilled water and purified powdered diets (7.5 g/mouse) in ceramic cups (10/group) from d 1-57 or 58, and were thereafter sacrificed in the morning by cervical dislocation over these last 2 experimental days. Food was not removed the night before, and since nocturnal mice are known to eat during the normal period dark cycle employed, the mice could have been fasted 0-6 h at sacrifice. Mice were thus not completely fasted at sacrifice, a complete fast being 12-16 h.  After feeding diets to 10 mice per group, 5 mice were randomly selected for inclusion in hippocampal gene expression analysis. From these same 5 mice, the hippocampus was dissected from the mice according to standard protocols (Porsolt & Partners Pharmacology, Boulogne-Billancourt, France), rinsed as described for livers, cut into 17-50 mg subsections, and frozen as described for livers.  Hippocampal lipid analysis was performed according to standard methods at Lipomics, Inc. (U.S.A) on individual mice (n=5).
Hippocampal tissue was extracted from 5 individual mice per group and extracted separately using Qiagen RNeasy mini-kits (Basel, Switzerland) according to manufacturer instructions, except that RNeasy columns were impregnated with DNase I (Roche, Basel, Switzerland) to remove any genomic DNA contamination. A pool of 10 mg total hippocampal RNA was created from the 5 individual extractions prior to GeneChip analysis. All RNA samples (individual and pooled) were quantified with the RiboGreen RNA Quantification Kit (Molecular Probes, Eugene Oregon), then analyzed via agarose gel electrophoresis for intact 18S and 28S rRNA. All study samples were judged to contain sufficient amounts of high-quality RNA for hybridization to GeneChips. As another quality control measure, prior to hybridization with Affymetrix GeneChips (Affymetrix, Inc., Santa Clara, CA), we confirmed that pooled samples gave strong signals using Affymetrix test chips.


title: 22-oct-2002-BrainEsubB-ab150

status: Public on Nov 11 2002

type: RNA

city: Lausanne

first: David

last: Vilanova

country: Switzerland

institute: Nestle Research Center

description:
Dietary effects of arachidonate-rich fungal oil and fish oil on murine hepatic and hippocampal gene expression, Lipids in Health and Disease 2002.
Diet contained 90% fat-free AIN93G rodent diet in powder form (Lot 9350-5, Dyets, Inc., Bethlehem, PA), 0.4% milk fat, 1.2% palm olein, 1.9% Trisun sunflower oil, 1.5% soybean oil and 2.1-5.1% medium chain triacylglycerol oil (all of the above ingredients except the AIN93G were from Nestlé affiliated companies). Part of the medium chain triacylglycerol oil in control diet was replaced with: 1.9% fish oil (providing 0.5 dietary wt% DHA and 1.0en%). Food was maintained at -80°C in daily aliquots under liquid N2, thawed each afternoon before administration to mice, and uneaten food was discarded daily.
Prior to actual experimentation, test dissections were made on identical mice to perfect the dissection techniques at Porsolt & Partners Pharmacology (Paris, France).
Male Rj:NMRI mice from Elevage Janvier, Le Genest-Saint-Isle France, weighing 10-11 g at delivery and 33-51 grams on experimental d 42, were housed 10 per cage in wire cages with bedding and normal light cycle. Mice received ad libitum quantities of distilled water and purified powdered diets (7.5 g/mouse) in ceramic cups (10/group) from d 1-57 or 58, and were thereafter sacrificed in the morning by cervical dislocation over these last 2 experimental days. Food was not removed the night before, and since nocturnal mice are known to eat during the normal period dark cycle employed, the mice could have been fasted 0-6 h at sacrifice. Mice were thus not completely fasted at sacrifice, a complete fast being 12-16 h.  After feeding diets to 10 mice per group, 5 mice were randomly selected for inclusion in hippocampal gene expression analysis. From these same 5 mice, the hippocampus was dissected from the mice according to standard protocols (Porsolt & Partners Pharmacology, Boulogne-Billancour, France) rinsed as described for livers, cut into 17-50 mg subsections, and frozen as described for livers.  Hippocampal lipid analysis was performed according to standard methods at Lipomics, Inc. (U.S.A) on individual mice (n=5).
Hippocampal tissue was extracted from 5 individual mice per group and extracted separately using Qiagen RNeasy mini-kits (Basel, Switzerland) according to manufacturer instructions, except that RNeasy columns were impregnated with DNase I (Roche, Basel, Switzerland) to remove any genomic DNA contamination. A pool of 10 mg total hippocampal RNA was created from the 5 individual extractions prior to GeneChip analysis. All RNA samples (individual and pooled) were quantified with the RiboGreen RNA Quantification Kit (Molecular Probes, Eugene Oregon), then analyzed via agarose gel electrophoresis for intact 18S and 28S rRNA. All study samples were judged to contain sufficient amounts of high-quality RNA for hybridization to GeneChips. As another quality control measure, prior to hybridization with Affymetrix GeneChips (Affymetrix, Inc., Santa Clara, CA), we confirmed that pooled samples gave strong signals using Affymetrix test chips.

molecule: total RNA

organism: Mus musculus

source_name: brain/hippocampus


title: 22-oct-2002-BrainDsubB-ab150


title: 22-oct-2002-liverA2-Mu11KsubAsc

status: Public on Nov 05 2002

description:
Dietary effects of arachidonate-rich fungal oil and fish oil on murine hepatic and hippocampal gene expression, Lipids in Health and Disease 2002.
Diet contained 90% fat-free AIN93G rodent diet in powder form (Lot 9350-5, Dyets, Inc., Bethlehem, PA), 0.4% milk fat, 1.2% palm olein, 1.9% Trisun sunflower oil, 1.5% soybean oil and 2.1-5.1% medium chain triacylglycerol oil (all of the above ingredients except the AIN93G were from Nestlé affiliated companies). Food was maintained at -80°C in daily aliquots under liquid N2, thawed each afternoon before administration to mice, and uneaten food was discarded daily.
Prior to actual experimentation, test dissections were made on identical mice to perfect the dissection techniques at Porsolt & Partners Pharmacology (Paris, France).
Male Rj:NMRI mice from Elevage Janvier, Le Genest-Saint-Isle France, weighing 10-11 g at delivery and 33-51 grams on experimental d 42, were housed 10 per cage in wire cages with bedding and normal light cycle. Mice received ad libitum quantities of distilled water and purified powdered diets (7.5 g/mouse) in ceramic cups (10/group) from d 1-57 or 58, and were thereafter sacrificed in the morning by cervical dislocation over these last 2 experimental days. Food was not removed the night before, and since nocturnal mice are known to eat during the normal period dark cycle employed, the mice could have been fasted 0-6 h at sacrifice. Mice were thus not completely fasted at sacrifice, a complete fast being 12-16 h.
After feeding diets to 10 mice per group, 5 mice were randomly selected for inclusion in hepatic gene expression analysis. From these 5 mice, livers were dissected according to standard protocols (Porsolt & Partners Pharmacology, Boulogne-Billancourt, France), which consisted of carefully dissecting out the liver, rinsing with 70% EtOH to remove adhering blood, trimming of adhering connective tissue with scissors, cutting into 100-150 mg subsections, freezing in liquid N2, and storing at -80°C until gene expression and lipid analysis. Hepatic lipid analysis was performed according to standard methods at Lipomics, Inc. (U.S.A) on individual mice (n=5).
Liver tissue was extracted from 5 individual mice per group and extracted separately using Qiagen RNeasy mini-kits (Basel, Switzerland) according to manufacturer instructions, except that RNeasy columns were impregnated with DNase I (Roche, Basel, Switzerland) to remove any genomic DNA contamination. A pool of 10 mg total hepatic RNA was created from the 5 individual extractions prior to GeneChip analysis. All RNA samples (individual and pooled) were quantified with the RiboGreen RNA Quantification Kit (Molecular Probes, Eugene Oregon), then analyzed via agarose gel electrophoresis for intact 18S and 28S rRNA. All study samples were judged to contain sufficient amounts of high-quality RNA for hybridization to GeneChips. As another quality control measure, prior to hybridization with Affymetrix GeneChips (Affymetrix, Inc., Santa Clara, CA), we confirmed that pooled samples gave strong signals using Affymetrix test chips.

source_name: liver/hepatocytes


title: 22-oct-2002-liverA1-Mu11KsubBsc


title: FHCMV-T-1

status: Public on Nov 08 2002

city: Seattle

first: Lynn

last: Graf

state: WA

address: 1100 Fairview Ave. N. D1-100

department: Clinical Research Division

country: USA

institute: Fred Hutchinson Cancer Research Center

description:
Target Source: human foreskin fibroblasts infected with cytomegalovirus
Description: Infection with clinical isolate FHCMV-T of CMV, genotype gB1. Overall comparison of gene expression profile of human foreskin fibroblasts (HFF) infected with 3 virus strains representing three glycoprotein B genotypes is described in (literature reference for manuscript).
Values are ratio of infected to uninfected fibroblasts.

HFF were plated in T75 flasks at approximately 80% confluence. The next day the supernatant was removed and replaced with the CMV viral inoculum (MOI of 3) in a total volume of 7 mls of RPMI supplemented with 5% FBS. The virus was permitted to absorb for 2 hours at 37 C and 5% CO2. The supernatants were removed from the non-infected control cells, replaced with 7 mls of RPMI, and incubated at the same time as the infected cultures. The inoculum was removed, the cells were washed 2 times with Hank's buffer, and media was replaced. Uninfected and infected fibroblasts were harvested at 24 hpi 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 42(C 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 uninfected and infected fibroblasts 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 table.

organism: Homo sapiens

source_name: FHCMV-T-infected fibroblasts

source_name: uninfected fibroblasts


title: CMV_AD169-3

description:
Target Source: human foreskin fibroblasts infected with cytomegalovirus
Description: Infection with AD169 strain of CMV, genotype gB1. Overall comparison of gene expression profile of human foreskin fibroblasts (HFF) infected with 3 virus strains representing three glycoprotein B genotypes is described in (literature reference for manuscript).
Values are ratio of infected to uninfected fibroblasts.

HFF were plated in T75 flasks at approximately 80% confluence. The next day the supernatant was removed and replaced with the CMV viral inoculum (MOI of 3) in a total volume of 7 mls of RPMI supplemented with 5% FBS. The virus was permitted to absorb for 2 hours at 37°C and 5% CO2. The supernatants were removed from the non-infected control cells, replaced with 7 mls of RPMI, and incubated at the same time as the infected cultures. The inoculum was removed, the cells were washed 2 times with Hank's buffer, and media was replaced. Uninfected and infected fibroblasts were harvested at 24 hpi 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 42°C 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 uninfected and infected fibroblasts 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 µl 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 table.

source_name: CMV AD169 infected fibroblasts


title: FHCMV-T-3

description:
Target Source: human foreskin fibroblasts infected with cytomegalovirus
Description: Infection with clinical isolate HCMV-T of CMV, genotype gB1. Overall comparison of gene expression profile of human foreskin fibroblasts (HFF) infected with 3 virus strains representing three glycoprotein B genotypes is described in (literature reference for manuscript).
Values are ratio of infected to uninfected fibroblasts.

HFF were plated in T75 flasks at approximately 80% confluence. The next day the supernatant was removed and replaced with the CMV viral inoculum (MOI of 3) in a total volume of 7 mls of RPMI supplemented with 5% FBS. The virus was permitted to absorb for 2 hours at 37°C and 5% CO2. The supernatants were removed from the non-infected control cells, replaced with 7 mls of RPMI, and incubated at the same time as the infected cultures. The inoculum was removed, the cells were washed 2 times with Hank's buffer, and media was replaced. Uninfected and infected fibroblasts were harvested at 24 hpi 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 42(C 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 uninfected and infected fibroblasts 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 µl 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 table.

source_name: CMV infected fibroblasts


title: SGearly08

status: Public on Jul 01 2002

city: Basel

first: Boris

last: Egger

state: BS

address: Klingelbergstrasse 50

department: University of Basel

institute: Institute of Zoology

description:
Experimental Design
To identify gcm downstream genes in a comprehensive manner, we used genome-wide oligonucleotide arrays to analyse differential gene expression in Drosophila wild-type embryos versus embryos in which gcm is misexpressed throughout the neuroectoderm.  The wild-type was Oregon-R.  Tissue-specific misexpression was achieved by using virgin females from scabrous-GAL4 (Klaes et al., 1994) that were crossed to yw; UAS-gcm; UAS-gcm males (Jones et al., 1995).  Transcripts were analysed at two defined temporal windows during embryogenesis.  First, during a period of initial gcm action on determination of glial cell precursors and second during a later period when glial cells have already differentiated.  After a 1 hr pre-collection, wild-type and sca-gcm embryos were collected in parallel for 1 hr and staged to 6-7 hrs AEL (stage 11) or to 13-14 hrs AEL (late stage 15/early stage 16).  Stages are according to (Campos-Ortega and Hartenstein, 1997).

Samples used, extract preparation and labeling
Total RNA was isolated from 200 mg of embryonic tissue, using guanidinium isothiocyanate in combination with acidic phenol (pH 4.0) (fast RNA tube green kit from BIO101) in a fast-prep homogenizer FP120 (BIO101).  After precipitation, the RNA was dissolved in DEPC-treated water (Ambion) and spectrophotometrically quantified using a GeneQuant RNA/DNA calculator (Pharmacia Biotech).  cDNA was synthesized upon total RNA as a template, using the SuperScript Choice System for cDNA synthesis (Gibco/BRL) with a T7-(T)24 DNA primer.  This primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24VN-3') was PAGE purified.  For first-strand cDNA synthesis, a typical 20 µl reaction contained 20 µg total RNA, 100 pmol T7-(T)24 primer, 1 mM of each dNTP and 800 units reverse transcriptase (AMV Superscript II).  The reaction was incubated for 1 hr at 42°C. Second-strand cDNA synthesis was carried out at 18°C for 2 hrs in a total volume of 150 µl, using 10 units E. coli DNA ligase, 40 units E. coli DNA polymerase I and 4 units Rnase A in the presence of 200 µM dNTPs.  After second-strand cDNA synthesis, samples were incubated with 20 units T4 DNA polymerase at 18°C for 5 min.  Then, 0.5 µl Rnase A 100 (µg/µl) (Quiagen) was added and the samples were incubated at 37°C for 30 min.  Thereafter, 2.5 µl proteinase K (30 mg/ml) (Sigma) was added and the samples were further incubated at 37°C for another 30 min.  After cDNA synthesis was completed, the samples were phenol-chloroform extracted using Phase Lock Gel (Eppendorf) and ethanol precipitated.  Biotinylated antisense cRNA was synthesized from the dsDNA template, using T7 RNA polymerase (MEGAscript T7 Kit, Ambion).  A 20 µl reaction volume contained between 0.3-1.5 µg cDNA, 7.5 mM of both ATP and GTP, 5.6 mM of both UTP and CTP and 1.8 mM of both biotinylated Bio-16-UTP and Bio-11-CTP (ENZO diagnostics) and 2µl 10x T7 enzyme mix.  The reaction was incubated at 37°C for 6-8 hrs.  Thereafter, the unincorporated dNTPs were removed by running the sample over an Rneasy spin column (Qiagen).  Samples were precipitated, dissolved in 20 µl DEPC-treated water and spectrophotometrically quantified.  Thereafter, 35 µg of the biotinylated antisense cRNA was fragmented by heating the sample to 95°C for 45 min in a volume of 25 µl, containing 40 mM tris-acetate (pH 8.1), 100 mM potassium acetate and 30 mM magnesium acetate.  After fragmentation, the samples were placed on ice.

Hybridization procedures
Gene Chips were prehybridized with 220 µl hybridization buffer ( 1x MES (pH 6.7), 1M NaCl, 0.01 % Tween 20, 0.5 µg/µl acetylated BSA, 0.5 µg/µl sonicated herring sperm DNA ) for 15 min at 40°C on a REAX 2 rotisserie (Heidolph, Swabach, Germany) at 60 rpm.  Hybridization was done in a final volume of 220 µl hybridization buffer containing 35 µg fragmented biotinylated cRNA.  The samples were heated to 95°C for 5 min and briefly spun down.  Hybridization was carried out for 16 hrs at 45°C on a rotisserie at 60 rpm.  After hybridization, the arrays were briefly rinsed with 6x SSPE-Tw (0.9 M NaCl, 0.06 M NaH2PO4, 6 mM EDTA, 0.01 % Tween 20) and washed on a Fluidics Station (Roche) for 5 min.  Chips were rinsed with MES-wash buffer (1x MES, 26 mM NaCl, 0.01 % Tween) and then 230 µl fresh MES-wash-buffer was applied to wash the chips at 45°C for 30 min with rotation.  Hybridized arrays were stained with 230 µl stain buffer (1xMES, 1M NaCl, with  2.3 µl streptavidin-R phycoerithrin conjugate (1 mg/ml) (Molecular Probes) and 2.5 µg/ml acetylated BSA (Sigma) at 40°C for 15 min and at 60 rpm.  The chips were rinsed and washed again on a Fluidics Station (Roche) for 5 min before scanning.

Measurement data and data processing
Probe arrays were scanned with a commercial confocal laser scanner (Hewlett-Packard). Pixel intensities were measured, and expression signals were analysed with commercial software (Genechip 4.0, Affymetrix).

organism: Drosophila melanogaster

source_name: sca-gcm embryos (6-7 hrs AEL)


title: CMV_AD169-2

source_name: CMV-AD169 infected fibroblasts


title: 22-oct-2002-liverF-Mu11KsubBsc

description:
Dietary effects of arachidonate-rich fungal oil and fish oil on murine hepatic and hippocampal gene expression, Lipids in Health and Disease 2002.
Diet contained 90% fat-free AIN93G rodent diet in powder form (Lot 9350-5, Dyets, Inc., Bethlehem, PA), 0.4% milk fat, 1.2% palm olein, 1.9% Trisun sunflower oil, 1.5% soybean oil and 2.1-5.1% medium chain triacylglycerol oil (all of the above ingredients except the AIN93G were from Nestlé affiliated companies). Part of the medium chain triacylglycerol oil in control diet was replaced with: 1.1% fungal oil and 1.9% fish oil. Food was maintained at -80°C in daily aliquots under liquid N2, thawed each afternoon before administration to mice, and uneaten food was discarded daily.
Prior to actual experimentation, test dissections were made on identical mice to perfect the dissection techniques at Porsolt & Partners Pharmacology (Paris, France).
Male Rj:NMRI mice from Elevage Janvier, Le Genest-Saint-Isle France, weighing 10-11 g at delivery and 33-51 grams on experimental d 42, were housed 10 per cage in wire cages with bedding and normal light cycle. Mice received ad libitum quantities of distilled water and purified powdered diets (7.5 g/mouse) in ceramic cups (10/group) from d 1-57 or 58, and were thereafter sacrificed in the morning by cervical dislocation over these last 2 experimental days. Food was not removed the night before, and since nocturnal mice are known to eat during the normal period dark cycle employed, the mice could have been fasted 0-6 h at sacrifice. Mice were thus not completely fasted at sacrifice, a complete fast being 12-16 h.
After feeding diets to 10 mice per group, 5 mice were randomly selected for inclusion in hepatic gene expression analysis. From these 5 mice, livers were dissected according to standard protocols (Porsolt & Partners Pharmacology, Boulogne-Billancourt, France), which consisted of carefully dissecting out the liver, rinsing with 70% EtOH to remove adhering blood, trimming of adhering connective tissue with scissors, cutting into 100-150 mg subsections, freezing in liquid N2, and storing at -80°C until gene expression and lipid analysis. Hepatic lipid analysis was performed according to standard methods at Lipomics, Inc. (U.S.A) on individual mice (n=5).
Liver tissue was extracted from 5 individual mice per group and extracted separately using Qiagen RNeasy mini-kits (Basel, Switzerland) according to manufacturer instructions, except that RNeasy columns were impregnated with DNase I (Roche, Basel, Switzerland) to remove any genomic DNA contamination. A pool of 10 mg total hepatic RNA was created from the 5 individual extractions prior to GeneChip analysis. All RNA samples (individual and pooled) were quantified with the RiboGreen RNA Quantification Kit (Molecular Probes, Eugene Oregon), then analyzed via agarose gel electrophoresis for intact 18S and 28S rRNA. All study samples were judged to contain sufficient amounts of high-quality RNA for hybridization to GeneChips. As another quality control measure, prior to hybridization with Affymetrix GeneChips (Affymetrix, Inc., Santa Clara, CA), we confirmed that pooled samples gave strong signals using Affymetrix test chips.


title: FHCMV-H-1

description:
Target Source: human foreskin fibroblasts infected with cytomegalovirus
Description: Infection with clinical isolate FHCMV-H of CMV, genotype gB3. Overall comparison of gene expression profile of human foreskin fibroblasts (HFF) infected with 3 virus strains representing three glycoprotein B genotypes is described in (literature reference for manuscript).
Values are ratio of infected to uninfected fibroblasts.

HFF were plated in T75 flasks at approximately 80% confluence. The next day the supernatant was removed and replaced with the CMV viral inoculum (MOI of 3) in a total volume of 7 mls of RPMI supplemented with 5% FBS. The virus was permitted to absorb for 2 hours at 37°C and 5% CO2. The supernatants were removed from the non-infected control cells, replaced with 7 mls of RPMI, and incubated at the same time as the infected cultures. The inoculum was removed, the cells were washed 2 times with Hank's buffer, and media was replaced. Uninfected and infected fibroblasts were harvested at 24 hpi 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 42°C 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 uninfected and infected fibroblasts 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 µl 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 table.


title: Sham3

status: Public on Aug 27 2002

city: St. Louis

first: Jason

middle: Matthew

last: Laramie

state: MO

address: 660 South Euclid Avenue

department: Surgery

institute: Washington University

description: C57/B6 mouse was subjected to sham laparotomy and sample taken 24hr after surgery.

source_name: Spleen


title: WTearly03

source_name: wild-type embryos (6-7 hrs AEL)


title: CF/B6 wild type 2mo ovary

status: Public on Jun 07 2002

city: Cleveland

first: John

middle: H

last: Nilson

state: OH

address: 2109 Adelbert Road

department: Pharmacology

institute: Case Western Reserve University, School of Medicine

description: Wild type ovary sample from 2 month old CF-1xC57BL/6 F1 hybrid mouse for use in gene expression profiling experiment on LH hypersecreting mice (LHCTP)

source_name: wild type ovary from CF-1xC57BL/6 F1 hybrid 2 month old mouse


title: CF/B6 transgenic 2mo ovary B

description: Transgenic, cystic ovary sample from 2 month old CF-1xC57BL/6 F1 hybrid mouse for use in gene expression profiling experiment on LH hypersecreting mice (LHCTP)

source_name: Transgenic ovary from 2 month old CF-1xC57BL/6 F1 hybrid mouse


title: CF/B6 transgenic 5mo ovary (luteoma) B

description: Transgenic (LHCTP), luteoma ovary sample from 5 month old CF-1xC57BL/6 F1 hybrid mouse for use in gene expression profiling experiment on LH hypersecreting mice (LHCTP)

source_name: Transgenic (LHCTP), luteoma ovary from 5 month old CF-1xC57BL/6 F1 hybrid mouse


title: WTearly02

source_name: wild-type embryos (6-7 hrs)


title: CF/B6 wild type 5mo ovary

description: Wild type ovary sample from 5 month old CF-1xC57BL/6 F1 hybrid mouse for use in gene expression profiling experiment on LH hypersecreting mice (LHCTP)

source_name: Wild type ovary from 5 month old CF-1xC57BL/6 F1 hybrid mouse


title: WTearly01

description:
Description
Experimental Design
To identify gcm downstream genes in a comprehensive manner, we used genome-wide oligonucleotide arrays to analyse differential gene expression in Drosophila wild-type embryos versus embryos in which gcm is misexpressed throughout the neuroectoderm.  The wild-type was Oregon-R.  Tissue-specific misexpression was achieved by using virgin females from scabrous-GAL4 (Klaes et al., 1994) that were crossed to yw; UAS-gcm; UAS-gcm males (Jones et al., 1995).  Transcripts were analysed at two defined temporal windows during embryogenesis.  First, during a period of initial gcm action on determination of glial cell precursors and second during a later period when glial cells have already differentiated.  After a 1 hr pre-collection, wild-type and sca-gcm embryos were collected in parallel for 1 hr and staged to 6-7 hrs AEL (stage 11) or to 13-14 hrs AEL (late stage 15/early stage 16).  Stages are according to (Campos-Ortega and Hartenstein, 1997).

Samples used, extract preparation and labeling
Total RNA was isolated from 200 mg of embryonic tissue, using guanidinium isothiocyanate in combination with acidic phenol (pH 4.0) (fast RNA tube green kit from BIO101) in a fast-prep homogenizer FP120 (BIO101).  After precipitation, the RNA was dissolved in DEPC-treated water (Ambion) and spectrophotometrically quantified using a GeneQuant RNA/DNA calculator (Pharmacia Biotech).  cDNA was synthesized upon total RNA as a template, using the SuperScript Choice System for cDNA synthesis (Gibco/BRL) with a T7-(T)24 DNA primer.  This primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24VN-3') was PAGE purified.  For first-strand cDNA synthesis, a typical 20 µl reaction contained 20 µg total RNA, 100 pmol T7-(T)24 primer, 1 mM of each dNTP and 800 units reverse transcriptase (AMV Superscript II).  The reaction was incubated for 1 hr at 42°C. Second-strand cDNA synthesis was carried out at 18°C for 2 hrs in a total volume of 150 µl, using 10 units E. coli DNA ligase, 40 units E. coli DNA polymerase I and 4 units Rnase A in the presence of 200 µM dNTPs.  After second-strand cDNA synthesis, samples were incubated with 20 units T4 DNA polymerase at 18°C for 5 min.  Then, 0.5 µl Rnase A 100 (µg/µl) (Quiagen) was added and the samples were incubated at 37°C for 30 min.  Thereafter, 2.5 µl proteinase K (30 mg/ml) (Sigma) was added and the samples were further incubated at 37°C for another 30 min.  After cDNA synthesis was completed, the samples were phenol-chloroform extracted using Phase Lock Gel (Eppendorf) and ethanol precipitated.  Biotinylated antisense cRNA was synthesized from the dsDNA template, using T7 RNA polymerase (MEGAscript T7 Kit, Ambion).  A 20 µl reaction volume contained between 0.3-1.5 µg cDNA, 7.5 mM of both ATP and GTP, 5.6 mM of both UTP and CTP and 1.8 mM of both biotinylated Bio-16-UTP and Bio-11-CTP (ENZO diagnostics) and 2µl 10x T7 enzyme mix.  The reaction was incubated at 37°C for 6-8 hrs.  Thereafter, the unincorporated dNTPs were removed by running the sample over an Rneasy spin column (Qiagen).  Samples were precipitated, dissolved in 20 µl DEPC-treated water and spectrophotometrically quantified.  Thereafter, 35 µg of the biotinylated antisense cRNA was fragmented by heating the sample to 95°C for 45 min in a volume of 25 µl, containing 40 mM tris-acetate (pH 8.1), 100 mM potassium acetate and 30 mM magnesium acetate.  After fragmentation, the samples were placed on ice.

Hybridization procedures
Gene Chips were prehybridized with 220 µl hybridization buffer ( 1x MES (pH 6.7), 1M NaCl, 0.01 % Tween 20, 0.5 µg/µl acetylated BSA, 0.5 µg/µl sonicated herring sperm DNA ) for 15 min at 40°C on a REAX 2 rotisserie (Heidolph, Swabach, Germany) at 60 rpm.  Hybridization was done in a final volume of 220 µl hybridization buffer containing 35 µg fragmented biotinylated cRNA.  The samples were heated to 95°C for 5 min and briefly spun down.  Hybridization was carried out for 16 hrs at 45°C on a rotisserie at 60 rpm.  After hybridization, the arrays were briefly rinsed with 6x SSPE-Tw (0.9 M NaCl, 0.06 M NaH2PO4, 6 mM EDTA, 0.01 % Tween 20) and washed on a Fluidics Station (Roche) for 5 min.  Chips were rinsed with MES-wash buffer (1x MES, 26 mM NaCl, 0.01 % Tween) and then 230 µl fresh MES-wash-buffer was applied to wash the chips at 45°C for 30 min with rotation.  Hybridized arrays were stained with 230 µl stain buffer (1xMES, 1M NaCl, with  2.3 µl streptavidin-R phycoerithrin conjugate (1 mg/ml) (Molecular Probes) and 2.5 µg/ml acetylated BSA (Sigma) at 40°C for 15 min and at 60 rpm.  The chips were rinsed and washed again on a Fluidics Station (Roche) for 5 min before scanning.

Measurement data and data processing
Probe arrays were scanned with a commercial confocal laser scanner (Hewlett-Packard). Pixel intensities were measured, and expression signals were analysed with commercial software (Genechip 4.0, Affymetrix).


title: CF/B6 transgenic 5mo ovary (luteoma)


title: SGearly10


title: CF/B6 wild type 5mo ovary B


title: HS-27a_05

status: Public on May 21 2002

first: Mineo

last: Iwata

department: Clinical Research

institute: Fred Hutchinson Cancer Research Institute

description:
Target Source: human bone marrow stromal cells, HS-27a
Description: human bone marrow stromal cells cultured in RPMI medium 1640 containing 10% fetal calf serum.
Values are ratio of HS-27a to Universal RNA.

HS-27a cells were plated in T75 flasks at 3,000,000 cells/flask. They were cultured for 3 days and harvested by trypsinization followed by pelleting of the cells. All RNA isolation was accomplished with Qiagen RNeasy Mini Kit  reagents. As a control RNA, Universal Human Reference RNA (Stratagene, La Jolla, CA) which is a mixture of RNA from 10 human cell lines approximating the expression profile of the majority of human genes was used. The RNA (30 µg) was annealed with 5 µg oligo dT12-18, and  reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42°C in the presence of  0.5 mM dGTP, 0.5 mM dCTP, 0.5 mM dATP, 0.3 mM dTTP, 0.2 mM amino-allyl dUTP. After hydrolysis of RNA in 0.2 M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from HS-27a RNA and Human Universal RNA was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 50 mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7 M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit and suspended in 36 µl 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 table.

source_name: HS-27a RNA

source_name: Universal human reference RNA


title: WTearly04

source_name: wild-type (6-7 hrs AEL)


title: FHCMV-H-3


title: CF-1 transgenic hCG Tx 5mo ovary B

description: Transgenic and hCG treated (luteoma) ovary sample from 5 month old CF-1 mouse for use in gene expression profiling experiment on LH hypersecreting mice (LHCTP)

source_name: Transgenic hCG treated ovary from 5 month old CF-1 mouse


title: 22-oct-2002-liverE-Mu11KsubAsc

description:
Dietary effects of arachidonate-rich fungal oil and fish oil on murine hepatic and hippocampal gene expression, Lipids in Health and Disease 2002.
Diet contained 90% fat-free AIN93G rodent diet in powder form (Lot 9350-5, Dyets, Inc., Bethlehem, PA), 0.4% milk fat, 1.2% palm olein, 1.9% Trisun sunflower oil, 1.5% soybean oil and 2.1-5.1% medium chain triacylglycerol oil (all of the above ingredients except the AIN93G were from Nestlé affiliated companies). Part of the medium chain triacylglycerol oil in control diet was replaced with: 1.9% fish oil (providing 0.5 dietary wt% DHA and 1.0en%). Food was maintained at -80°C in daily aliquots under liquid N2, thawed each afternoon before administration to mice, and uneaten food was discarded daily.
Prior to actual experimentation, test dissections were made on identical mice to perfect the dissection techniques at Porsolt & Partners Pharmacology (Paris, France).
Male Rj:NMRI mice from Elevage Janvier, Le Genest-Saint-Isle France, weighing 10-11 g at delivery and 33-51 grams on experimental d 42, were housed 10 per cage in wire cages with bedding and normal light cycle. Mice received ad libitum quantities of distilled water and purified powdered diets (7.5 g/mouse) in ceramic cups (10/group) from d 1-57 or 58, and were thereafter sacrificed in the morning by cervical dislocation over these last 2 experimental days. Food was not removed the night before, and since nocturnal mice are known to eat during the normal period dark cycle employed, the mice could have been fasted 0-6 h at sacrifice. Mice were thus not completely fasted at sacrifice, a complete fast being 12-16 h.
After feeding diets to 10 mice per group, 5 mice were randomly selected for inclusion in hepatic gene expression analysis. From these 5 mice, livers were dissected according to standard protocols (Porsolt & Partners Pharmacology, Boulogne-Billancourt, France), which consisted of carefully dissecting out the liver, rinsing with 70% EtOH to remove adhering blood, trimming of adhering connective tissue with scissors, cutting into 100-150 mg subsections, freezing in liquid N2, and storing at -80°C until gene expression and lipid analysis. Hepatic lipid analysis was performed according to standard methods at Lipomics, Inc. (U.S.A) on individual mice (n=5).
Liver tissue was extracted from 5 individual mice per group and extracted separately using Qiagen RNeasy mini-kits (Basel, Switzerland) according to manufacturer instructions, except that RNeasy columns were impregnated with DNase I (Roche, Basel, Switzerland) to remove any genomic DNA contamination. A pool of 10 mg total hepatic RNA was created from the 5 individual extractions prior to GeneChip analysis. All RNA samples (individual and pooled) were quantified with the RiboGreen RNA Quantification Kit (Molecular Probes, Eugene Oregon), then analyzed via agarose gel electrophoresis for intact 18S and 28S rRNA. All study samples were judged to contain sufficient amounts of high-quality RNA for hybridization to GeneChips. As another quality control measure, prior to hybridization with Affymetrix GeneChips (Affymetrix, Inc., Santa Clara, CA), we confirmed that pooled samples gave strong signals using Affymetrix test chips.

source_name: liver/hepatocytes


title: HS-5_06

description:
Target Source: human bone marrow stromal cells, HS-5
Description: human bone marrow stromal cells cultured in RPMI medium 1640 containing 10% fetal calf serum.
Values are ratio of HS-5 to Universal RNA.

HS-5 cells were plated in T75 flasks at 3,000,000 cells/flask. They were cultured for 3 days and harvested by trypsinization followed by pelleting of the cells. All RNA isolation was accomplished with Qiagen RNeasy Mini Kit  reagents. As a control RNA, Human Universal RNA (Stratagene, La Jolla, CA) which is a mixture of RNA from 10 control cell lines approximating the expression profile of the majority of human genes was used. The RNA (30 µg) was annealed with 5 µg oligo dT12-18, and  reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42°C in the presence of  0.5 mM dGTP, 0.5 mM dCTP, 0.5  mM dATP, 0.3 mM dTTP, 0.2 mM amino-allyl dUTP. After hydrolysis of RNA in 0.2 M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from HS-5 RNA and Human Universal RNA was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 50 mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7 M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit and suspended in 36 µl 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 table.

source_name: HS-5 RNA


title: hswt_3

status: Public on Feb 25 2002

first: Thomas

last: Loop

department: Institute of Zoology

institute: University of Basel

description:
The wildtype used was Drosophila melanogaster Oregon-R. All fly stocks were kept on standard cornmeal/yeast/agar medium at 25°C.  Embryos were collected overnight for 12 hours on grape juice plates, further kept for 4 hours at 25°C and then subjected to a 37°C heat shock for 25 min, followed by a recovery period of 25 min at 25°C before RNA isolation.  Therefore, at the time of RNA isolation these embryos were at embryonic stages 10-17 [Leemans et al.: Quantitative transcript imaging in normal and heat-shocked Drosophila embryos by using high-density oligonucleotide arrays. Proc Natl Acad Sci U S A 2000; 97:12138-12143.]. Embryos younger than embryonic stage 10 were not used, since heat shock in these earlier stages results in lethality [Walter set al.: Heat shock causes the collapse of the intermediate filament cytoskeleton in Drosophila embryos. Dev Genet 1990; 11:270-279.]

Total RNA was isolated from 200 mg of embryonic tissue, using guanidinium isothiocyanate in combination with acidic phenol (pH 4.0) (fast RNA tube green kit from BIO101) in a fast prep homogenizer FP120 (BIO 101). After precipitation, the RNA was dissolved in DEPC-treated water (Ambion) and spectrophotometrically quantified using a GeneQuant RNA/DNA calculator (Pharmacia Biotech). cDNA was synthesized upon total RNA as a template, using the SuperScript Choice System for cDNA synthesis (Gibco/BRL) with a T7-(T)24 DNA primer.
This primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24VN-3') was purified by PAGE. For first-strand cDNA synthesis, a typical 40 ul reaction contained 25 ug RNA, 200 pmol T7-(T)24 primer, 500 uM of each dNTP and 800 units reverse transcriptase (AMV Superscript II). The reaction was incubated for 1 h at 42°C. Second-strand cDNA synthesis was carried out at 18°C for 2 h in a total volume of 340 ul, using 20 units Escherichia coli DNA ligase, 80 units E. coli DNA polymerase I and 4 units RNase H in the presence of 250 uM of each dNTP. After second-strand cDNA synthesis, 0.5 ul RNase A (100 mg/ml) (Qiagen) was added and the samples were incubated at 37°C for 30 min. Thereafter, 7.5 ul proteinase K (10 mg/ml) (Sigma) was added and the samples were further incubated at 37°C for another 30 min. After cDNA synthesis was completed, samples were phenol-chloroform extracted, using Phase Lock Gel (5 Prime-3 Prime) and ethanol precipitated. Biotinylated antisense cRNA was synthesized from the dsDNA template, using T7 RNA polymerase (MEGAscript T7 Kit: Ambion.). A 20 ul reaction volume contained between 0.3-1.5 ug cDNA, 7.5 mM of both ATP and GTP, 5.6 mM of both UTP and CTP and 1.8 mM of both biotinylated Bio-16-UTP and Bio-11-CTP (ENZO diagnostics) and 2 ul 10x T7 enzyme mix. The reaction was incubated at 37°C for 8 h. Thereafter, the unincorporated NTPs were removed by running the sample over an RNeasy spin column (Qiagen). Samples were precipitated, taken up in 20 ul DEPC-treated water and spectrophotometrically quantified. Thereafter, 40 ug of the biotinylated antisense cRNA was fragmented by heating the sample to 95°C for 35 min in a volume of 25 ul, containing 40 mM tris-acetate (pH 8.1), 100 mM potassium acetate, 30 mM magnesium acetate. After the fragmentation, the samples were placed on ice.
Gene Chips were pre-hybridized with 220 ul hybridization buffer (1x MES (pH 6.7), 1 M NaCl, 0.01% Triton, 0.5 g/l acetylated BSA, 0.5 g/l sonicated herring sperm DNA) for 15 min at 45°C on a rotisserie (Heidolph) at 60 rpm. Hybridization was done in a final volume of 220 ul hybridization buffer, containing 40 g fragmented biotinylated cRNA. The samples were heated to 95°C for 5 min and briefly spun down. Hybridizations were carried out for 16 h at 45°C with mixing on a rotisserie at 60 rpm. After hybridization, the arrays were briefly rinsed with 6x SSPE-T (0.9 M NaCl, 0.06 M NaH2PO4, 6 mM EDTA, 0.01% Triton) and washed on a Fluidics station (Affymetrix). Hybridized arrays were stained with 220 ul detection solution (1x MES buffer, containing 2.5 ul streptavidin-R phycoerythrin conjugate (1 mg/ml) (Molecular Probes)) and 2.0 mg/ml acetylated BSA (Sigma) at 40°C for 15 min and washed again before being scanned.

The data was normalized against the mean of the total sums of Avg Diff values across all 16 arrays used in this study.

source_name: Drosophila melanogaster embryos


title: CF-1 transgenic 5mo ovary (granulosa cell tumor)

description: Transgenic (LHCTP), granulosa cell tumor sample from ovary from 5 month old CF-1 mouse for use in gene expression profiling experiment on LH hypersecreting mice (LHCTP)

source_name: Transgenic (LHCTP) ovary from 5 month old CF-1 mouse


title: hswt_2


title: wt_5

description:
The wildtype used was Drosophila melanogaster Oregon-R. All fly stocks were kept on standard cornmeal/yeast/agar medium at 25°C.  At the time of RNA isolation these embryos were at embryonic stages 10-17 [Leemans et al.: Quantitative transcript imaging in normal and heat-shocked Drosophila embryos by using high-density oligonucleotide arrays. Proc Natl Acad Sci U S A 2000; 97:12138-12143.]. Embryos younger than embryonic stage 10 were not used.

Total RNA was isolated from 200 mg of embryonic tissue, using guanidinium isothiocyanate in combination with acidic phenol (pH 4.0) (fast RNA tube green kit from BIO101) in a fast prep homogenizer FP120 (BIO 101). After precipitation, the RNA was dissolved in DEPC-treated water (Ambion) and spectrophotometrically quantified using a GeneQuant RNA/DNA calculator (Pharmacia Biotech). cDNA was synthesized upon total RNA as a template, using the SuperScript Choice System for cDNA synthesis (Gibco/BRL) with a T7-(T)24 DNA primer.
This primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24VN-3') was purified by PAGE. For first-strand cDNA synthesis, a typical 40 ul reaction contained 25 ug RNA, 200 pmol T7-(T)24 primer, 500 uM of each dNTP and 800 units reverse transcriptase (AMV Superscript II). The reaction was incubated for 1 h at 42°C. Second-strand cDNA synthesis was carried out at 18°C for 2 h in a total volume of 340 ul, using 20 units Escherichia coli DNA ligase, 80 units E. coli DNA polymerase I and 4 units RNase H in the presence of 250 uM of each dNTP. After second-strand cDNA synthesis, 0.5 ul RNase A (100 mg/ml) (Qiagen) was added and the samples were incubated at 37°C for 30 min. Thereafter, 7.5 ul proteinase K (10 mg/ml) (Sigma) was added and the samples were further incubated at 37°C for another 30 min. After cDNA synthesis was completed, samples were phenol-chloroform extracted, using Phase Lock Gel (5 Prime-3 Prime) and ethanol precipitated. Biotinylated antisense cRNA was synthesized from the dsDNA template, using T7 RNA polymerase (MEGAscript T7 Kit: Ambion.). A 20 ul reaction volume contained between 0.3-1.5 ug cDNA, 7.5 mM of both ATP and GTP, 5.6 mM of both UTP and CTP and 1.8 mM of both biotinylated Bio-16-UTP and Bio-11-CTP (ENZO diagnostics) and 2 ul 10x T7 enzyme mix. The reaction was incubated at 37°C for 8 h. Thereafter, the unincorporated NTPs were removed by running the sample over an RNeasy spin column (Qiagen). Samples were precipitated, taken up in 20 ul DEPC-treated water and spectrophotometrically quantified. Thereafter, 40 ug of the biotinylated antisense cRNA was fragmented by heating the sample to 95°C for 35 min in a volume of 25 ul, containing 40 mM tris-acetate (pH 8.1), 100 mM potassium acetate, 30 mM magnesium acetate. After the fragmentation, the samples were placed on ice.
Gene Chips were pre-hybridized with 220 ul hybridization buffer (1x MES (pH 6.7), 1 M NaCl, 0.01% Triton, 0.5 g/l acetylated BSA, 0.5 g/l sonicated herring sperm DNA) for 15 min at 45°C on a rotisserie (Heidolph) at 60 rpm. Hybridization was done in a final volume of 220 ul hybridization buffer, containing 40 g fragmented biotinylated cRNA. The samples were heated to 95°C for 5 min and briefly spun down. Hybridizations were carried out for 16 h at 45°C with mixing on a rotisserie at 60 rpm. After hybridization, the arrays were briefly rinsed with 6x SSPE-T (0.9 M NaCl, 0.06 M NaH2PO4, 6 mM EDTA, 0.01% Triton) and washed on a Fluidics station (Affymetrix). Hybridized arrays were stained with 220 ul detection solution (1x MES buffer, containing 2.5 ul streptavidin-R phycoerythrin conjugate (1 mg/ml) (Molecular Probes)) and 2.0 mg/ml acetylated BSA (Sigma) at 40°C for 15 min and washed again before being scanned.

The data was normalized against the mean of the total sums of Avg Diff values across all 16 arrays used in this study.


title: wt_2


title: wt_3


title: HS-27a_27


title: CF-1 transgenic 2mo ovary

description: Transgenic (LHCTP), cystic ovary sample from 2 month old CF-1 mouse for use in gene expression profiling experiment on LH hypersecreting mice (LHCTP)

source_name: LHCTP transgenic ovary from 2 month old CF-1 mouse


title: HS-5_04

description:
Target Source: human bone marrow stromal cells, HS-5
Description: human bone marrow stromal cells cultured in RPMI medium 1640 containing 10% fetal calf serum.
Values are ratio of HS-5 to Universal RNA.

HS-5 cells were plated in T75 flasks at 3,000,000 cells/flask. They were cultured for 3 days and harvested by trypsinization followed by pelleting of the cells. All RNA isolation was accomplished with Qiagen RNeasy Mini Kit  reagents. As a control RNA, Human Universal RNA (Stratagene, La Jolla, CA) which is a mixture of RNA from 10 control cell lines approximating the expression profile of the majority of human genes was used. The RNA (30 µg) was annealed with 5 µg oligo dT12-18, and  reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42°C in the presence of  0.5 mM dGTP, 0.5 mM dCTP, 0.5 mM dATP, 0.3 mM dTTP, 0.2 mM amino-allyl dUTP. After hydrolysis of RNA in 0.2 M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from HS-5 RNA and Human Universal RNA was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 50 mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7 M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit and suspended in 36 µl 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 table.


title: CF1 transgenic 2mo ovary B

source_name: Transgenic (LHCTP) ovary from 2 month old CF-1 mouse chip B


title: HS-5_05


title: CF-1 wild type 5mo ovary B

description: Wild type ovary sample from 5 month old CF-1 mouse for use in gene expression profiling experiment on LH hypersecreting mice (LHCTP)

source_name: Wild type ovary from 5 month old CF-1 mouse


title: wt_1


title: wt_4


title: hsOTX2_4

description:
For overexpression of human Otx2, the hsp-Otx2 line generated by Leuzinger et al. was used [Leuzinger et al.: Equivalence of the fly orthodenticle gene and the human OTX genes in embryonic brain development of Drosophila. Development 1998; 125:1703-1710.]. All fly stocks were kept on standard
cornmeal/yeast/agar medium at 25°C.  Embryos were collected overnight for 12 hours on grape juice plates, further kept for 4 hours at 25°C and then subjected to a 37°C heat shock for 25 min, followed by a recovery period of 25 min at 25°C before RNA isolation.  Therefore, at the time of RNA isolation these embryos were at embryonic stages 10-17 [Leemans et al.: Quantitative transcript imaging in normal and heat-shocked Drosophila embryos by using high-density oligonucleotide arrays. Proc Natl Acad Sci U S A 2000; 97:12138-12143.]. Embryos younger than embryonic stage 10 were not used, since heat shock in these earlier stages results in lethality [Walter set al.: Heat shock causes the collapse of the intermediate filament cytoskeleton in Drosophila embryos. Dev Genet 1990; 11:270-279.]

Total RNA was isolated from 200 mg of embryonic tissue, using guanidinium isothiocyanate in combination with acidic phenol (pH 4.0) (fast RNA tube green kit from BIO101) in a fast prep homogenizer FP120 (BIO 101). After precipitation, the RNA was dissolved in DEPC-treated water (Ambion) and spectrophotometrically quantified using a GeneQuant RNA/DNA calculator (Pharmacia Biotech). cDNA was synthesized upon total RNA as a template, using the SuperScript Choice System for cDNA synthesis (Gibco/BRL) with a T7-(T)24 DNA primer.
This primer (5'-GCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24VN-3') was purified by PAGE. For first-strand cDNA synthesis, a typical 40 ul reaction contained 25 ug RNA, 200 pmol T7-(T)24 primer, 500 M of each dNTP and 800 units reverse transcriptase (AMV Superscript II). The reaction was incubated for 1 h at 42°C. Second-strand cDNA synthesis was carried out at 18°C for 2 h in a total volume of 340 ul, using 20 units Escherichia coli DNA ligase, 80 units E. coli DNA polymerase I and 4 units RNase H in the presence of 250 M of each dNTP. After second-strand cDNA synthesis, 0.5 ul RNase A (100 mg/ml)(Qiagen) was added and the samples were incubated at 37°C for 30 min. Thereafter, 7.5 ul proteinase K (10 mg/ml) (Sigma) was added and the samples were further incubated at 37°C for another 30 min. After cDNA synthesis was completed, samples were phenol-chloroform extracted, using Phase Lock Gel (5 Prime-3 Prime) and ethanol precipitated. Biotinylated antisense cRNA was synthesized from the dsDNA template, using T7 RNA polymerase (MEGAscript T7 Kit: Ambion.). A 20 l reaction volume contained between 0.3-1.5 g cDNA, 7.5 mM of both ATP and GTP, 5.6 mM of both UTP and CTP and 1.8 mM of both biotinylated Bio-16-UTP and Bio-11-CTP (ENZO diagnostics) and 2 ul 10x T7 enzyme mix. The reaction was incubated at 37°C for 8 h. Thereafter, the unincorporated NTPs were removed by running the sample over an RNeasy spin column (Qiagen). Samples were precipitated, taken up in 20 ul DEPC-treated water and spectrophotometrically quantified. Thereafter, 40 ug of the biotinylated antisense cRNA was fragmented by heating the sample to 95°C for 35 min in a volume of 25 ul, containing 40 mM tris-acetate (pH 8.1), 100 mM potassium acetate, 30 mM magnesium acetate. After the fragmentation, the samples were placed on ice.
Gene Chips were pre-hybridized with 220 ul hybridization buffer (1x MES (pH 6.7), 1 M NaCl, 0.01% Triton, 0.5 g/l acetylated BSA, 0.5 g/l sonicated herring sperm DNA) for 15 min at 45°C on a rotisserie (Heidolph) at 60 rpm. Hybridization was done in a final volume of 220 ul hybridization buffer, containing 40 g fragmented biotinylated cRNA. The samples were heated to 95°C for 5 min and briefly spun down. Hybridizations were carried out for 16 h at 45°C with mixing on a rotisserie at 60 rpm. After hybridization, the arrays were briefly rinsed with 6x SSPE-T (0.9 M NaCl, 0.06 M NaH2PO4, 6 mM EDTA, 0.01% Triton) and washed on a Fluidics station (Affymetrix). Hybridized arrays were stained with 220 ul detection solution (1x MES buffer, containing 2.5 l streptavidin-R phycoerythrin conjugate (1 mg/ml) (Molecular Probes)) and 2.0 mg/ml acetylated BSA (Sigma) at 40°C for 15 min and washed again.

The data was normalized against the mean of the total sums of Avg Diff values across all 16 arrays used in this study.


title: CF-1 wild type 5mo ovary

source_name: wild type ovary from 5 month CF-1 mouse


title: Mature B cells 11

status: Public on Jan 09 2002

city: Munich

first: Reinhard

last: Hoffmann

address: Trogerstr. 30

country: Germany

institute: Institute for medical microbiology

description: B220hi sIgM+ Mature B cells sorted out of mouse bone marrow, sort no. 11

source_name: sorted Murine Bone marrow cells


title: Small Pre-BII cells 8b

status: Public on Dec 17 2001

description: B220+CD25+sIg- Small Pre BII cells sorted out of mouse bone marrow, sort no. 8

source_name: Small Pre-BII cells


title: hswt_1


title: hsOTX2_3

description:
For overexpression of human Otx2, the hsp-Otx2 line generated by Leuzinger et al. was used [Leuzinger et al.: Equivalence of the fly orthodenticle gene and the human OTX genes in embryonic brain development of Drosophila. Development 1998; 125:1703-1710.]. All fly stocks were kept on standard
cornmeal/yeast/agar medium at 25°C.  Embryos were collected overnight for 12 hours on grape juice plates, further kept for 4 hours at 25°C and then subjected to a 37°C heat shock for 25 min, followed by a recovery period of 25 min at 25°C before RNA isolation.  Therefore, at the time of RNA isolation these embryos were at embryonic stages 10-17 [Leemans et al.: Quantitative transcript imaging in normal and heat-shocked Drosophila embryos by using high-density oligonucleotide arrays. Proc Natl Acad Sci U S A 2000; 97:12138-12143.]. Embryos younger than embryonic stage 10 were not used, since heat shock in these earlier stages results in lethality [Walter set al.: Heat shock causes the collapse of the intermediate filament cytoskeleton in Drosophila embryos. Dev Genet 1990; 11:270-279.]

Total RNA was isolated from 200 mg of embryonic tissue, using guanidinium isothiocyanate in combination with acidic phenol (pH 4.0) (fast RNA tube green kit from BIO101) in a fast prep homogenizer FP120 (BIO 101). After precipitation, the RNA was dissolved in DEPC-treated water (Ambion) and spectrophotometrically quantified using a GeneQuant RNA/DNA calculator (Pharmacia Biotech). cDNA was synthesized upon total RNA as a template, using the SuperScript Choice System for cDNA synthesis (Gibco/BRL) with a T7-(T)24 DNA primer.
This primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24VN-3') was purified by PAGE. For first-strand cDNA synthesis, a typical 40 ul reaction contained 25 ug RNA, 200 pmol T7-(T)24 primer, 500 uM of each dNTP and 800 units reverse transcriptase (AMV Superscript II). The reaction was incubated for 1 h at 42°C. Second-strand cDNA synthesis was carried out at 18°C for 2 h in a total volume of 340 ul, using 20 units Escherichia coli DNA ligase, 80 units E. coli DNA polymerase I and 4 units RNase H in the presence of 250 uM of each dNTP. After second-strand cDNA synthesis, 0.5 ul RNase A (100 mg/ml) (Qiagen) was added and the samples were incubated at 37°C for 30 min. Thereafter, 7.5 ul proteinase K (10 mg/ml) (Sigma) was added and the samples were further incubated at 37°C for another 30 min. After cDNA synthesis was completed, samples were phenol-chloroform extracted, using Phase Lock Gel (5 Prime-3 Prime) and ethanol precipitated. Biotinylated antisense cRNA was synthesized from the dsDNA template, using T7 RNA polymerase (MEGAscript T7 Kit: Ambion.). A 20 ul reaction volume contained between 0.3-1.5 ug cDNA, 7.5 mM of both ATP and GTP, 5.6 mM of both UTP and CTP and 1.8 mM of both biotinylated Bio-16-UTP and Bio-11-CTP (ENZO diagnostics) and 2 ul 10x T7 enzyme mix. The reaction was incubated at 37°C for 8 h. Thereafter, the unincorporated NTPs were removed by running the sample over an RNeasy spin column (Qiagen). Samples were precipitated, taken up in 20 ul DEPC-treated water and spectrophotometrically quantified. Thereafter, 40 ug of the biotinylated antisense cRNA was fragmented by heating the sample to 95°C for 35 min in a volume of 25 ul, containing 40 mM tris-acetate (pH 8.1), 100 mM potassium acetate, 30 mM magnesium acetate. After the fragmentation, the samples were placed on ice.
Gene Chips were pre-hybridized with 220 ul hybridization buffer (1x MES (pH 6.7), 1 M NaCl, 0.01% Triton, 0.5 g/l acetylated BSA, 0.5 g/l sonicated herring sperm DNA) for 15 min at 45°C on a rotisserie (Heidolph) at 60 rpm. Hybridization was done in a final volume of 220 ul hybridization buffer, containing 40 g fragmented biotinylated cRNA. The samples were heated to 95°C for 5 min and briefly spun down. Hybridizations were carried out for 16 h at 45°C with mixing on a rotisserie at 60 rpm. After hybridization, the arrays were briefly rinsed with 6x SSPE-T (0.9 M NaCl, 0.06 M NaH2PO4, 6 mM EDTA, 0.01% Triton) and washed on a Fluidics station (Affymetrix). Hybridized arrays were stained with 220 ul detection solution (1x MES buffer, containing 2.5 ul streptavidin-R phycoerythrin conjugate (1 mg/ml) (Molecular Probes)) and 2.0 mg/ml acetylated BSA (Sigma) at 40°C for 15 min and washed again before being scanned.

The data was normalized against the mean of the total sums of Avg Diff values across all 16 arrays used in this study.


title: hsOTX2_1


title: Small Pre-BII cells 3

description: B220+CD25+sIg- Small Pre BII cells sorted out of mouse bone marrow, sort no. 3


title: hsOTX2_2


title: Small Pre-BII cells 8


title: 22-oct-2002-BrainEsubA-ab150

description:
Dietary effects of arachidonate-rich fungal oil and fish oil on murine hepatic and hippocampal gene expression, Lipids in Health and Disease 2002.
Diet contained 90% fat-free AIN93G rodent diet in powder form (Lot 9350-5, Dyets, Inc., Bethlehem, PA), 0.4% milk fat, 1.2% palm olein, 1.9% Trisun sunflower oil, 1.5% soybean oil and 2.1-5.1% medium chain triacylglycerol oil (all of the above ingredients except the AIN93G were from Nestlé affiliated companies). Part of the medium chain triacylglycerol oil in control diet was replaced with: 1.9% fish oil (providing 0.5 dietary wt% DHA and 1.0en%). Food was maintained at -80°C in daily aliquots under liquid N2, thawed each afternoon before administration to mice, and uneaten food was discarded daily.
Prior to actual experimentation, test dissections were made on identical mice to perfect the dissection techniques at Porsolt & Partners Pharmacology (Paris, France).
Male Rj:NMRI mice from Elevage Janvier, Le Genest-Saint-Isle France, weighing 10-11 g at delivery and 33-51 grams on experimental d 42, were housed 10 per cage in wire cages with bedding and normal light cycle. Mice received ad libitum quantities of distilled water and purified powdered diets (7.5 g/mouse) in ceramic cups (10/group) from d 1-57 or 58, and were thereafter sacrificed in the morning by cervical dislocation over these last 2 experimental days. Food was not removed the night before, and since nocturnal mice are known to eat during the normal period dark cycle employed, the mice could have been fasted 0-6 h at sacrifice. Mice were thus not completely fasted at sacrifice, a complete fast being 12-16 h.  After feeding diets to 10 mice per group, 5 mice were randomly selected for inclusion in hippocampal gene expression analysis. From these same 5 mice, the hippocampus was dissected from the mice according to standard protocols (Porsolt & Partners Pharmacology, Boulogne-Billancourt, France), rinsed as described for livers, cut into 17-50 mg subsections, and frozen as described for livers.  Hippocampal lipid analysis was performed according to standard methods at Lipomics, Inc. (U.S.A) on individual mice (n=5).
Hippocampal tissue was extracted from 5 individual mice per group and extracted separately using Qiagen RNeasy mini-kits (Basel, Switzerland) according to manufacturer instructions, except that RNeasy columns were impregnated with DNase I (Roche, Basel, Switzerland) to remove any genomic DNA contamination. A pool of 10 mg total hippocampal RNA was created from the 5 individual extractions prior to GeneChip analysis. All RNA samples (individual and pooled) were quantified with the RiboGreen RNA Quantification Kit (Molecular Probes, Eugene Oregon), then analyzed via agarose gel electrophoresis for intact 18S and 28S rRNA. All study samples were judged to contain sufficient amounts of high-quality RNA for hybridization to GeneChips. As another quality control measure, prior to hybridization with Affymetrix GeneChips (Affymetrix, Inc., Santa Clara, CA), we confirmed that pooled samples gave strong signals using Affymetrix test chips.

source_name: brain/hippocampus


title: SGearly07

description:
Experimental Design
To identify gcm downstream genes in a comprehensive manner, we used genome-wide oligonucleotide arrays to analyse differential gene expression in Drosophila wild-type embryos versus embryos in which gcm is misexpressed throughout the neuroectoderm.  The wild-type was Oregon-R.  Tissue-specific misexpression was achieved by using virgin females from scabrous-GAL4 (Klaes et al., 1994) that were crossed to yw; UAS-gcm; UAS-gcm males (Jones et al., 1995).  Transcripts were analysed at two defined temporal windows during embryogenesis.  First, during a period of initial gcm action on determination of glial cell precursors and second during a later period when glial cells have already differentiated.  After a 1 hr pre-collection, wild-type and sca-gcm embryos were collected in parallel for 1 hr and staged to 6-7 hrs AEL (stage 11) or to 13-14 hrs AEL (late stage 15/early stage 16).  Stages are according to (Campos-Ortega and Hartenstein, 1997).

Samples used, extract preparation and labeling
Total RNA was isolated from 200 mg of embryonic tissue, using guanidinium isothiocyanate in combination with acidic phenol (pH 4.0) (fast RNA tube green kit from BIO101) in a fast-prep homogenizer FP120 (BIO101).  After precipitation, the RNA was dissolved in DEPC-treated water (Ambion) and spectrophotometrically quantified using a GeneQuant RNA/DNA calculator (Pharmacia Biotech).  cDNA was synthesized upon total RNA as a template, using the SuperScript Choice System for cDNA synthesis (Gibco/BRL) with a T7-(T)24 DNA primer.  This primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24VN-3') was PAGE purified.  For first-strand cDNA synthesis, a typical 20 µl reaction contained 20 µg total RNA, 100 pmol T7-(T)24 primer, 1 mM of each dNTP and 800 units reverse transcriptase (AMV Superscript II).  The reaction was incubated for 1 hr at 42°C. Second-strand cDNA synthesis was carried out at 18°C for 2 hrs in a total volume of 150 µl, using 10 units E. coli DNA ligase, 40 units E. coli DNA polymerase I and 4 units Rnase A in the presence of 200 µM dNTPs.  After second-strand cDNA synthesis, samples were incubated with 20 units T4 DNA polymerase at 18°C for 5 min.  Then, 0.5 µl Rnase A 100 (µg/µl) (Quiagen) was added and the samples were incubated at 37°C for 30 min.  Thereafter, 2.5 µl proteinase K (30 mg/ml) (Sigma) was added and the samples were further incubated at 37°C for another 30 min.  After cDNA synthesis was completed, the samples were phenol-chloroform extracted using Phase Lock Gel (Eppendorf) and ethanol precipitated.  Biotinylated antisense cRNA was synthesized from the dsDNA template, using T7 RNA polymerase (MEGAscript T7 Kit, Ambion).  A 20 µl reaction volume contained between 0.3-1.5 µg cDNA, 7.5 mM of both ATP and GTP, 5.6 mM of both UTP and CTP and 1.8 mM of both biotinylated Bio-16-UTP and Bio-11-CTP (ENZO diagnostics) and 2µl 10x T7 enzyme mix.  The reaction was incubated at 37°C for 6-8 hrs.  Thereafter, the unincorporated dNTPs were removed by running the sample over an Rneasy spin column (Qiagen).  Samples were precipitated, dissolved in 20 µl DEPC-treated water and spectrophotometrically quantified.  Thereafter, 35 µg of the biotinylated antisense cRNA was fragmented by heating the sample to 95°C for 45 min in a volume of 25 µl, containing 40 mM tris-acetate (pH 8.1), 100 mM potassium acetate and 30 mM magnesium acetate.  After fragmentation, the samples were placed on ice.

Hybridization procedures
Gene Chips were prehybridized with 220 µl hybridization buffer ( 1x MES (pH 6.7), 1M NaCl, 0.01 % Tween 20, 0.5 µg/µl acetylated BSA, 0.5 µg/µl sonicated herring sperm DNA ) for 15 min at 40°C on a REAX 2 rotisserie (Heidolph, Swabach, Germany) at 60 rpm.  Hybridization was done in a final volume of 220 µl hybridization buffer containing 35 µg fragmented biotinylated cRNA.  The samples were heated to 95°C for 5 min and briefly spun down.  Hybridization was carried out for 16 hrs at 45°C on a rotisserie at 60 rpm.  After hybridization, the arrays were briefly rinsed with 6x SSPE-Tw (0.9 M NaCl, 0.06 M NaH2PO4, 6 mM EDTA, 0.01 % Tween 20) and washed on a Fluidics Station (Roche) for 5 min.  Chips were rinsed with MES-wash buffer (1x MES, 26 mM NaCl, 0.01 % Tween) and then 230 µl fresh MES-wash-buffer was applied to wash the chips at 45°C for 30 min with rotation.  Hybridized arrays were stained with 230 µl stain buffer (1xMES, 1M NaCl, with  2.3 µl streptavidin-R phycoerithrin conjugate (1 mg/ml) (Molecular Probes) and 2.5 µg/ml acetylated BSA (Sigma) at 40°C for 15 min and at 60 rpm.  The chips were rinsed and washed again on a Fluidics Station (Roche) for 5 min before scanning.

Measurement data and data processing
Probe arrays were scanned with a commercial confocal laser scanner (Hewlett-Packard). Pixel intensities were measured, and expression signals were analysed with commercial software (Genechip 4.0, Affymetrix).

source_name: sca-gcm embryos (6-7 hrs AEL)


title: Small Pre-BII cells 3b


title: Pre-BI cells 2

description: B220+c-kit+ Pre BI cells sorted out of mouse bone marrow, sort no. 2

source_name: Pre-BI cells


title: Pre-BI cells 10

description: B220+c-kit+ Pre BI cells sorted out of mouse bone marrow, sort no. 10


title: Small Pre-BII cells 10b

description: B220+CD25+sIg- Small Pre BII cells sorted out of mouse bone marrow, sort no. 10


title: Pre-BI cells 9b

description: B220+c-kit+ Pre BI cells sorted out of mouse bone marrow, sort no. 9


title: Small Pre-BII cells 10


title: hsotd_2

description:
For overexpression of otd, the hsp-otd line 5A generated by Royet and Finkelstein was used [Royet et al.: Pattern formation in Drosophila head development: the role of the orthodenticle homeobox gene. Development 1995; 121:3561-3572.]. All fly stocks were kept on standard cornmeal/yeast/agar medium at 25°C.  Embryos were collected overnight for 12 hours on grape juice plates, further kept for 4 hours at 25°C and then subjected to a 37°C heat shock for 25 min, followed by a recovery period of 25 min at 25°C before RNA isolation.  Therefore, at the time of RNA isolation these embryos were at embryonic stages 10-17 [Leemans et al.: Quantitative transcript imaging in normal and heat-shocked Drosophila embryos by using high-density oligonucleotide arrays. Proc Natl Acad Sci U S A 2000; 97:12138-12143.]. Embryos younger than embryonic stage 10 were not used, since heat shock in these earlier stages results in lethality [Walter set al.: Heat shock causes the collapse of the intermediate filament cytoskeleton in Drosophila embryos. Dev Genet 1990; 11:270-279.]

Total RNA was isolated from 200 mg of embryonic tissue, using guanidinium isothiocyanate in combination with acidic phenol (pH 4.0) (fast RNA tube green kit from BIO101) in a fast prep homogenizer FP120 (BIO 101). After precipitation, the RNA was dissolved in DEPC-treated water (Ambion) and spectrophotometrically quantified using a GeneQuant RNA/DNA calculator (Pharmacia Biotech). cDNA was synthesized upon total RNA as a template, using the SuperScript Choice System for cDNA synthesis (Gibco/BRL) with a T7-(T)24 DNA primer.
This primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24VN-3') was purified by PAGE. For first-strand cDNA synthesis, a typical 40 ul reaction contained 25 ug RNA, 200 pmol T7-(T)24 primer, 500 uM of each dNTP and 800 units reverse transcriptase (AMV Superscript II). The reaction was incubated for 1 h at 42°C. Second-strand cDNA synthesis was carried out at 18°C for 2 h in a total volume of 340 ul, using 20 units Escherichia coli DNA ligase, 80 units E. coli DNA polymerase I and 4 units RNase H in the presence of 250 uM of each dNTP. After second-strand cDNA synthesis, 0.5 ul RNase A (100 mg/ml) (Qiagen) was added and the samples were incubated at 37°C for 30 min. Thereafter, 7.5 ul proteinase K (10 mg/ml) (Sigma) was added and the samples were further incubated at 37°C for another 30 min. After cDNA synthesis was completed, samples were phenol-chloroform extracted, using Phase Lock Gel (5 Prime-3 Prime) and ethanol precipitated. Biotinylated antisense cRNA was synthesized from the dsDNA template, using T7 RNA polymerase (MEGAscript T7 Kit: Ambion.). A 20 ul reaction volume contained between 0.3-1.5 ug cDNA, 7.5 mM of both ATP and GTP, 5.6 mM of both UTP and CTP and 1.8 mM of both biotinylated Bio-16-UTP and Bio-11-CTP (ENZO diagnostics) and 2 ul 10x T7 enzyme mix. The reaction was incubated at 37°C for 8 h. Thereafter, the unincorporated NTPs were removed by running the sample over an RNeasy spin column (Qiagen). Samples were precipitated, taken up in 20 ul DEPC-treated water and spectrophotometrically quantified. Thereafter, 40 ug of the biotinylated antisense cRNA was fragmented by heating the sample to 95°C for 35 min in a volume of 25 ul, containing 40 mM tris-acetate (pH 8.1), 100 mM potassium acetate, 30 mM magnesium acetate. After the fragmentation, the samples were placed on ice.
Gene Chips were pre-hybridized with 220 ul hybridization buffer (1x MES (pH 6.7), 1 M NaCl, 0.01% Triton, 0.5 g/l acetylated BSA, 0.5 g/l sonicated herring sperm DNA) for 15 min at 45°C on a rotisserie (Heidolph) at 60 rpm. Hybridization was done in a final volume of 220 ul hybridization buffer, containing 40 g fragmented biotinylated cRNA. The samples were heated to 95°C for 5 min and briefly spun down. Hybridizations were carried out for 16 h at 45°C with mixing on a rotisserie at 60 rpm. After hybridization, the arrays were briefly rinsed with 6x SSPE-T (0.9 M NaCl, 0.06 M NaH2PO4, 6 mM EDTA, 0.01% Triton) and washed on a Fluidics station (Affymetrix). Hybridized arrays were stained with 220 ul detection solution (1x MES buffer, containing 2.5 ul streptavidin-R phycoerythrin conjugate (1 mg/ml) (Molecular Probes)) and 2.0 mg/ml acetylated BSA (Sigma) at 40°C for 15 min and washed again before being scanned.

The data was normalized against the mean of the total sums of Avg Diff values across all 16 arrays used in this study.


title: Mature B cells 3

description: B220hi sIgM+ Mature B cells sorted out of mouse bone marrow, sort no. 3

source_name: Mature B cells


title: Small Pre-BII cells 1b

description: B220+CD25+sIg- Small Pre BII cells sorted out of mouse bone marrow, sort no. 1


title: Pre-BI cells 3b

description: B220+c-kit+ Pre BI cells sorted out of mouse bone marrow, sort no. 3


title: hsotd_3


title: Pre-BI cells 9


title: Pre-BI cells 8

description: B220+c-kit+ Pre BI cells sorted out of mouse bone marrow, sort no. 8


title: Pre-BI cells 8b


title: Mature B cells 1_2

description: B220hi sIgM+ Mature B cells sorted out of mouse bone marrow, sort no. 1_2


title: Pre-BI cells 3

description: B220+c-kit+ Pre BIcells sorted out of mouse bone marrow, sort no. 3


title: Pre-BI cells 1b

description: B220+c-kit+ Pre BI cells sorted out of mouse bone marrow, sort no. 1


title: Large Pre-BII cells 8

description: B220+CD25+sIg- Large Pre BII sorted out of mouse bone marrow, sort no. 8

source_name: Large Pre-BII cells


title: Large Pre-BII cells 1_2

description: B220+CD25+sIg- Large Pre BII cells sorted out of mouse bone marrow, sort no. 1_2


title: Mature B cells 3-2b

description: B220hi sIgM+ Mature B cells sorted out of mouse bone marrow, sort no. MAT3-2


title: Mature B cells 9b

description: B220hi sIgM+ Mature B cells sorted out of mouse bone marrow, sort no. 9


title: Mature B cells 8

description: B220hi sIgM+ Mature B cells sorted out of mouse bone marrow, sort no. 8


title: Immature B cells 9

description: B220Low sIgM+ immature B cells sorted out of mouse bone marrow, sort no. 9

source_name: Immature B cells


title: Large Pre-BII cells 10

description: B220+CD25+sIg- Large Pre BII cells sorted out of mouse bone marrow, sort no. 10


title: Large Pre-BII cells 9

description: B220+CD25+sIg- Large Pre BII cells sorted out of mouse bone marrow, sort no. 9


title: Large Pre-BII cells 2

description: B220+CD25+sIg- Large Pre BII cells sorted out of mouse bone marrow, sort no. 2


title: Immature B cells 2

description: B220Low sIgM+ immature B cells sorted out of mouse bone marrow, sort no. 2


title: Large Pre-BII cells 10b


title: Immature B cells 8

description: B220Low sIgM+ immature B cells sorted out of mouse bone marrow, sort no. 8


title: Pre-BI cells 10b

description: B220+c-kit+ Pre BIcells sorted out of mouse bone marrow, sort no. 10


title: Immature B cells 11

description: B220Low sIgM+ immature B cells sorted out of mouse bone marrow, sort no. 11


title: Large Pre-BII cells 8b

description: B220+CD25+sIg- Large Pre BII cells sorted out of mouse bone marrow, sort no. 8


title: Immature B cells 8b


title: Mature B cells 11b

description: B220hi sIgM+ Mature B cells sorted out of mouse bone marrow, sort no. 11


title: Large Pre-BII cells 9b


title: Immature B cells 9b


title: Mature B cells 1b

description: B220hi sIgM+ Mature B cells sorted out of mouse bone marrow, sort no. 1


title: Immature B cells 1

description: Immature B cells cells sorted out of mouse bone marrow, sort no. 1


title: Large Pre-BII cells 1b

description: B220+CD25+sIg- Large Pre BII cells sorted out of mouse bone marrow, sort no. 1


title: SGearly09

description:
Experimental Design
To identify gcm downstream genes in a comprehensive manner, we used genome-wide oligonucleotide arrays to analyse differential gene expression in Drosophila wild-type embryos versus embryos in which gcm is misexpressed throughout the neuroectoderm.  The wild-type was Oregon-R.  Tissue-specific misexpression was achieved by using virgin females from scabrous-GAL4 (Klaes et al., 1994) that were crossed to yw; UAS-gcm; UAS-gcm males (Jones et al., 1995).  Transcripts were analysed at two defined temporal windows during embryogenesis.  First, during a period of initial gcm action on determination of glial cell precursors and second during a later period when glial cells have already differentiated.  After a 1 hr pre-collection, wild-type and sca-gcm embryos were collected in parallel for 1 hr and staged to 6-7 hrs AEL (stage 11) or to 13-14 hrs AEL (late stage 15/early stage 16).  Stages are according to (Campos-Ortega and Hartenstein, 1997).

Samples used, extract preparation and labeling
Total RNA was isolated from 200 mg of embryonic tissue, using guanidinium isothiocyanate in combination with acidic phenol (pH 4.0) (fast RNA tube green kit from BIO101) in a fast-prep homogenizer FP120 (BIO101).  After precipitation, the RNA was dissolved in DEPC-treated water (Ambion) and spectrophotometrically quantified using a GeneQuant RNA/DNA calculator (Pharmacia Biotech).  cDNA was synthesized upon total RNA as a template, using the SuperScript Choice System for cDNA synthesis (Gibco/BRL) with a T7-(T)24 DNA primer.  This primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T)24VN-3') was PAGE purified.  For first-strand cDNA synthesis, a typical 20 µl reaction contained 20 µg total RNA, 100 pmol T7-(T)24 primer, 1 mM of each dNTP and 800 units reverse transcriptase (AMV Superscript II).  The reaction was incubated for 1 hr at 42°C. Second-strand cDNA synthesis was carried out at 18°C for 2 hrs in a total volume of 150 µl, using 10 units E. coli DNA ligase, 40 units E. coli DNA polymerase I and 4 units Rnase A in the presence of 200 µM dNTPs.  After second-strand cDNA synthesis, samples were incubated with 20 units T4 DNA polymerase at 18°C for 5 min.  Then, 0.5 µl Rnase A 100 (µg/µl) (Quiagen) was added and the samples were incubated at 37°C for 30 min.  Thereafter, 2.5 µl proteinase K (30 mg/ml) (Sigma) was added and the samples were further incubated at 37°C for another 30 min.  After cDNA synthesis was completed, the samples were phenol-chloroform extracted using Phase Lock Gel (Eppendorf) and ethanol precipitated.  Biotinylated antisense cRNA was synthesized from the dsDNA template, using T7 RNA polymerase (MEGAscript T7 Kit, Ambion).  A 20 µl reaction volume contained between 0.3-1.5 µg cDNA, 7.5 mM of both ATP and GTP, 5.6 mM of both UTP and CTP and 1.8 mM of both biotinylated Bio-16-UTP and Bio-11-CTP (ENZO diagnostics) and 2µl 10x T7 enzyme mix.  The reaction was incubated at 37°C for 6-8 hrs.  Thereafter, the unincorporated dNTPs were removed by running the sample over an Rneasy spin column (Qiagen).  Samples were precipitated, dissolved in 20 µl DEPC-treated water and spectrophotometrically quantified.  Thereafter, 35 µg of the biotinylated antisense cRNA was fragmented by heating the sample to 95°C for 45 min in a volume of 25 µl, containing 40 mM tris-acetate (pH 8.1), 100 mM potassium acetate and 30 mM magnesium acetate.  After fragmentation, the samples were placed on ice.

Hybridization procedures
Gene Chips were prehybridized with 220 µl hybridization buffer ( 1x MES (pH 6.7), 1M NaCl, 0.01 % Tween 20, 0.5 µg/µl acetylated BSA, 0.5 µg/µl sonicated herring sperm DNA ) for 15 min at 40°C on a REAX 2 rotisserie (Heidolph, Swabach, Germany) at 60 rpm.  Hybridization was done in a final volume of 220 µl hybridization buffer containing 35 µg fragmented biotinylated cRNA.  The samples were heated to 95°C for 5 min and briefly spun down.  Hybridization was carried out for 16 hrs at 45°C on a rotisserie at 60 rpm.  After hybridization, the arrays were briefly rinsed with 6x SSPE-Tw (0.9 M NaCl, 0.06 M NaH2PO4, 6 mM EDTA, 0.01 % Tween 20) and washed on a Fluidics Station (Roche) for 5 min.  Chips were rinsed with MES-wash buffer (1x MES, 26 mM NaCl, 0.01 % Tween) and then 230 µl fresh MES-wash-buffer was applied to wash the chips at 45°C for 30 min with rotation.  Hybridized arrays were stained with 230 µl stain buffer (1xMES, 1M NaCl, with  2.3 µl streptavidin-R phycoerithrin conjugate (1 mg/ml) (Molecular Probes) and 2.5 µg/ml acetylated BSA (Sigma) at 40°C for 15 min and at 60 rpm.  The chips were rinsed and washed again on a Fluidics Station (Roche) for 5 min before scanning.

Measurement data and data processing
Probe arrays were scanned with a commercial confocal laser scanner (Hewlett-Packard). Pixel intensities were measured, and expression signals were analysed with commercial software (Genechip 4.0, Affymetrix).


title: Large Pre-BII cells 2b


title: Immature B cells 1b

description: B220Low sIgM+ immature B cells sorted out of mouse bone marrow, sort no. 1


title: CF/B6 transgenic 2mo ovary

description: Transgenic, cystic ovary sample from 2 month old CF-1xC57BL/6 F1 hybrid mouse for use in gene expression profiling experiment on LH hypersecreting mice (LHCTP)

source_name: Transgenic (LHCTP) ovary from 2 month old CF-1xC57BL/6 F1 hybrid mouse


title: female b

status: Public on Oct 18 2000

city: Bethesda

first: Brian

last: Oliver

state: MD

address: 50 South Drive

department: LBG

institute: NIDDK, NIH

description: Whole adult male minus (12-24 hours post-eclosion) Drosophila melanogaster of the genotype y w[67c1].

source_name: y w[67c1] female


title: FHCMV-T-2

description:
Target Source: human foreskin fibroblasts infected with cytomegalovirus
Infection with clinical isolate FHCMV-T of CMV, genotype gB1. Overall comparison of gene expression profile of human foreskin fibroblasts (HFF) infected with 3 virus strains representing two glycoprotein B genotypes is described in (literature reference for manuscript).
Values are ratio of infected to uninfected fibroblasts.

HFF were plated in T75 flasks at approximately 80% confluence. The next day the supernatant was removed and replaced with the CMV viral inoculum (MOI of 3) in a total volume of 7 mls of RPMI supplemented with 5% FBS. The virus was permitted to absorb for 2 hours at 37 C and 5% CO2. The supernatants were removed from the non-infected control cells, replaced with 7 mls of RPMI, and incubated at the same time as the infected cultures. The inoculum was removed, the cells were washed 2 times with Hank's buffer, and media was replaced. Uninfected and infected fibroblasts were harvested at 24 hpi 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 42(C 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 uninfected and infected fibroblasts 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 table.

source_name: CMV infected fibroblasts

source_name: uninfected fibroblasts


title: Thalamus_Forward

status: Public on Mar 24 2006

city: Urbana

first: Mark

middle: Robert

last: Band

state: IL

address: 1201 W. Gregory Dr

department: Biotechnology

institute: University of Illinois

description: Samples were labeled with an indirect labeling protocol using amino-allyl dUTP and hybridized overnight at 42C

data_processing: Value = log2 ratio of background corrected 632 median/background corrected 532 median

organism: Bos taurus

source_name: Thalamus 635

tag: Male, 2 week old Jersey calf

source_name: Universal Control 532

tag: Total RNA from bovine cell cultures of :

tag: Brain

tag: EBTR (tracheal cell line)

tag: MDBK (kidney cell line)

tag: BL-30 (lymphocyte line)