title: Differentiated Subcutaneous Replicate 1

description:
Trizol isolation of total RNA from Differentiated Human Subcutaneous Preadipocytes.
Subject: Caucasian Female, 31 YO, BMI 47.9, Fasting Glucose 114 mg/dl.
Fat tissue was resected during gastric bypass surgery for management of obesity.  All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hank’s balanced salt solution with bicarbonate, penicillin, and gentamicin.  Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer.  Cells were plated in 1:1 Dulbecco’s modified Eagle’s medium:Ham’s F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 M rosiglitazone, antibiotics, and 540 M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days.  For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol.  RNA was isolated from preadipocytes by the Trizol method.  RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis.  Samples were hybridized for 16 hours at 45 ºC and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm.  Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Microarray Summary Statistics: Scaling Factor 3.500, Corner + 74, Corner -  74, Background 61.11, Noise 2.85, Raw Q 2.53
Keywords = adipocyte, subcutaneous, mesenteric, omental


title: MR vs sed (M6-64)

status: Public on May 26 2005

type: RNA

city: Baton Rouge

first: Leslie

middle: DeBardeleben

last: McLaughlin

state: LA

address: 6400 Perkins Road

department: Department of Experimental Obesity

country: USA

institute: Pennington Biomedical Research Center

description:
Source1 (Channel 1; Cy5)= Sedentary controls (6 male C5lBl/6J mice); pooled RNA from the hippocampus of 6 sed (no running).

Source2 (Channel 2; Cy3)= Middle Runners (6 male C57Bl/6J mice); pooled RNA from the hippocampus of 6 MR (ran 2-5 km/night).

Total RNA was extracted from the frozen hippocampi of individual animals using TRI REAGENT® (Molecular Research Center, Inc, Cincinnati, OH), followed by purification on Qiagen columns from the RNAeasy kit. Equal amounts (6-mg) of total RNA from each of the randomly chosen middle (n=6) MR and sedentary controls (n=6) were combined to generate 2 separate pools. Pooled total RNA was primed with oligo(dT) for reverse transcription, labeled with Cy3 and Cy5 dyes using the Micromax TSA Labeling and Detection Kit protocol (Perkin Elmer Life Sciences, Inc., Boston, MA) and hybridized to the in-house spotted slides. Six microarrays were hybridized (3 forward-labeled, 3 reverse-labeled, to account for differential dye incorporation) per comparison. The protocol for labeling and hybridization was basically as follows. The differentially fluorescent-labeled first-strand cDNA from each source was suspended in 22 µl of hybridization solution containing 5µl of poly-A (5mg/ml), 12.5µl of 20x SSC, 0.5µl 10% SDS and 12.5µl deionized formamide, combined and hybridized to the arrays for 30-36 hrs. The signals from specifically hybridized biotin- and fluorescein-labeled cDNAs were amplified either with streptavidin-horseradish peroxidase (HRP) and Cy5-tyramide or antifluorescein-HRP and Cy3-tyramide, respectively. Fluorescent array images were collected with a GSI Lumonics ScanArray 5000 fluorescent scanner (Packard Biochip Technologies; Meriden, CT) and image intensity data were extracted and analyzed using the provided QuantArray V. 3.0 software (Packard Bioscience; Meriden, CT). A uniform scale factor was applied to normalize signal intensities between Cy5 and Cy3. LOWESS normalization was applied to account for dye variation across and between slides, and for signal intensity effects. An in-house software program was used to perform this analysis (Details of the algorithm can be found at http://bioinfo.pbrc.edu). Fluorescent intensities below 500 were removed, and the expression ratios were LOWESS transformed to obtain the log base 2 values given in the data table.

Keywords = hippocampus
Keywords = voluntary running
Keywords = C57Bl/6J
Lot batch = PBRC M6-64

molecule: total RNA

organism: Mus musculus

source_name: pooled hippocampal RNA from sedentary controls

source_name: pooled hippocampal RNA from MR


title: sed vs HR (M6-48)

description:
Source2 (Channel 2; Cy3)= Sedentary controls (6 male C5lBl/6J mice); pooled RNA from the hippocampus of 6 sed (no running).

Source1 (Channel 1; Cy5)= High Runners (6 male C57Bl/6J mice); pooled RNA from the hippocampus of 6 HR (ran >= 5km/night).

Total RNA was extracted from the frozen hippocampi of individual animals using TRI REAGENT® (Molecular Research Center, Inc, Cincinnati, OH), followed by purification on Qiagen columns from the RNAeasy kit. Equal amounts (6-mg) of total RNA from each of the top (n=6) HR and sedentary controls (n=6) were combined to generate 2 separate pools. Pooled total RNA was primed with oligo(dT) for reverse transcription, labeled with Cy3 and Cy5 dyes using the Micromax TSA Labeling and Detection Kit protocol (Perkin Elmer Life Sciences, Inc., Boston, MA) and hybridized to the in-house spotted slides. Six microarrays were hybridized (3 forward-labeled, 3 reverse-labeled, to account for differential dye incorporation) per comparison. The protocol for labeling and hybridization was basically as follows. The differentially fluorescent-labeled first-strand cDNA from each source was suspended in 22 µl of hybridization solution containing 5µl of poly-A (5mg/ml), 12.5µl of 20x SSC, 0.5µl 10% SDS and 12.5µl deionized formamide, combined and hybridized to the arrays for 30-36 hrs. The signals from specifically hybridized biotin- and fluorescein-labeled cDNAs were amplified either with streptavidin-horseradish peroxidase (HRP) and Cy5-tyramide or antifluorescein-HRP and Cy3-tyramide, respectively. Fluorescent array images were collected with a GSI Lumonics ScanArray 5000 fluorescent scanner (Packard Biochip Technologies; Meriden, CT) and image intensity data were extracted and analyzed using the provided QuantArray V. 3.0 software (Packard Bioscience; Meriden, CT). A uniform scale factor was applied to normalize signal intensities between Cy5 and Cy3. LOWESS normalization was applied to account for dye variation across and between slides, and for signal intensity effects. An in-house software program was used to perform this analysis (Details of the algorithm can be found at http://bioinfo.pbrc.edu). Fluorescent intensities below 500 were removed, and the expression ratios were LOWESS transformed to obtain the log base 2 values given in the data table.

Lot batch = PBRC M6-48

source_name: pooled hippocampal RNA from HR


title: HR vs sed (M6-45)

description:
Source1 (Channel 1; Cy3)= High Runners (6 male C57Bl/6J mice); pooled RNA from the hippocampus of 6 HR (ran >= 5km/night).

Source2 (Channel 2; Cy5)= Sedentary controls (6 male C5lBl/6J mice); pooled RNA from the hippocampus of 6 sed (no running).

Total RNA was extracted from the frozen hippocampi of individual animals using TRI REAGENT® (Molecular Research Center, Inc, Cincinnati, OH), followed by purification on Qiagen columns from the RNAeasy kit. Equal amounts (6-mg) of total RNA from each of the top (n=6) HR and sedentary controls (n=6) were combined to generate 2 separate pools. Pooled total RNA was primed with oligo(dT) for reverse transcription, labeled with Cy3 and Cy5 dyes using the Micromax TSA Labeling and Detection Kit protocol (Perkin Elmer Life Sciences, Inc., Boston, MA) and hybridized to the in-house spotted slides. Six microarrays were hybridized (3 forward-labeled, 3 reverse-labeled, to account for differential dye incorporation) per comparison. The protocol for labeling and hybridization was basically as follows. The differentially fluorescent-labeled first-strand cDNA from each source was suspended in 22 µl of hybridization solution containing 5µl of poly-A (5mg/ml), 12.5µl of 20x SSC, 0.5µl 10% SDS and 12.5µl deionized formamide, combined and hybridized to the arrays for 30-36 hrs. The signals from specifically hybridized biotin- and fluorescein-labeled cDNAs were amplified either with streptavidin-horseradish peroxidase (HRP) and Cy5-tyramide or antifluorescein-HRP and Cy3-tyramide, respectively. Fluorescent array images were collected with a GSI Lumonics ScanArray 5000 fluorescent scanner (Packard Biochip Technologies; Meriden, CT) and image intensity data were extracted and analyzed using the provided QuantArray V. 3.0 software (Packard Bioscience; Meriden, CT). A uniform scale factor was applied to normalize signal intensities between Cy5 and Cy3. LOWESS normalization was applied to account for dye variation across and between slides, and for signal intensity effects. An in-house software program was used to perform this analysis (Details of the algorithm can be found at http://bioinfo.pbrc.edu). Fluorescent intensities below 500 were removed, and the expression ratios were LOWESS transformed to obtain the log base 2 values given in the data table.

Keywords = hippocampus
Keywords = voluntary running
Keywords = C57Bl/6J
Lot batch = PBRC M6-45


title: det2_Mock3

status: Public on May 24 2005

city: Stanford

first: Joshua

middle: Martin

last: Gendron

state: CA

address: 260 Panama Rd.

department: Plant Biology

institute: Carnegie Institution of Washington/Stanford University

description:
Growth Conditions and Treatments: Arabidopsis thaliana seeds were surface sterilized. Approximately 15000 seeds(300 milligrams) were placed in a 1 L erlenmeyer flasks containing 300 mL of liquid Murashige and Skoog medium (Sigma, St Louis, MO, USA) containing 1.5% sucrose. These flasks were incubated with gentle shaking for 5 days under constant illumination at 22ºC. After this time, 15 ul of 80% ethanol was added as the mock control (BL treated received 15 ul of 2uM Brassinolide (BL) dissolved in 80% ethanol.  The BL treatment results were not used and are thus not reported). Flasks were incubated for 3 hours with the ethanol. Whole seedlings from each treatment were collected, the medium was drained, and the seedlings were rapidly forzen in liquid nitrogen. The experiment was done alongside two other genetic backgrounds (wildtype and bzr1-1D) and was repeated three times (experiments 1-3) during different weeks.
RNA and Microarray Methods: Total RNA was extracted from the plants using a modified Trizol method (Chomczynski and Sashi, 1987)(see also Protocols Manual at the AFGC web page http://www.arabidopsis.org/info/2010_projects/comp_proj/AFGC/RevisedAFGC/AFGC_Protocols_Dec_2001L.pdf) and purified with a silica membrane column (Qiagen, RNeasy). Fifteen micrograms biotinylated complementary RNA (cRNA) was prepared and used to hybridize ATH1 Arabidopsis GeneChips (Affymetrix) using the manufacturer’s protocols. The array images were analyzed with the Affymetrix GeneChip Operating Software (GCOS) 1.1 with the target intensity set to 500. These data were imported into GeneSpring 7.0. To remove chip-to-chip and experiment-to-experiment signal variation each array was normalized by the intensity values of the top 50th percentile of genes on this array. The individual detection values were then normalized to the corresponding detection values of the wildtype (Colombia-0) control for each experiment. The normalized values for the data set are reported along with the intensity values for this array.
Keywords = Arabidopsis, hormone, brassinosteroid, det2, bzr1-1D
Lot batch = 510690

organism: Arabidopsis thaliana

source_name: Genotype: Columbia-0, det2 mut.; Treatment: incubated with 80 percent ethanol for 3 hours; Biological Replicate: 3 of three


title: Normal Peritoneal Macrophages/Neutrophils

status: Public on May 31 2005

city: Los Angeles

first: Adrian

middle: Friedrich

last: Gombart

institute: Cedars-Sinai Medical Center

description:
Experimental Design
Prior studies of C/EBPe-deficient myeloid cells from both humans and mice, demonstrated an essential role for C/EBPe in the normal development and function of both neutrophils and macrophages. There are striking parallels between human neutrophil specific granule disease (SGD) due to mutations in the CEBPE gene and the murine C/EBPe-deficient condition.  This indicates that the C/EBPe-deficient murine model will serve as an extremely powerful tool in further characterizing this rare human disease.  In this study, we sought to define further the role that C/EBPe plays in mediating host immune function by identifying possible target genes of C/EBPe in both neutrophils and macrophages.  This study was designed to elucidate the effects of the CEBPE gene knockout in murine myeloid cells.

Mice and sample preparation
The C/EBPE wild type (+/+) and –deficient (-/-) mice (129/SvEv x NIH Black Swiss) were generously provided by K.G. Xanthopoulos (Anadys Pharmaceuticals, Inc., San Diego, CA) and Julie Lekstrom-Himes (Millennium Pharmaceuticals, Inc., Cambridge, MA).  They were maintained in pathogen-free facilities on normal chow.  To prepare RNA for array hybridization, four age-matched (6-8 weeks) mice of each genotype received an intraperitoneal (IP) injection of 2 ml of 4% sterile thioglycollate broth (Sigma Chemical Co., St Louis, MO).  At 24 h post injection, all mice were sacrificed and peritoneal exudate cells were harvested by lavage with Hanks’ balanced salt solution (HBSS; Sigma Chemical Co.) and kept on ice.  Total cell numbers were counted and percentages of neutrophils and macrophages were determined by differential counting of Wright-Giemsa stained cytospins (100-200 cells per sample) using a light microscope.   The preparations were composed of approximately 30% monocytes, 60% neutrophils and 7-10% lymphocytes in both the C/EBPe+/+ (wt) and C/EBPe-/- (ko) mice.  To reduce individual variation in subsequent experiments, the cells either from all wild type or all C/EBPe-null mice were pooled prior to probe synthesis (n=1 pooled RNA for wt and n=1 pooled RNA for ko)
Total RNA and protein was isolated from cells using TRIzol reagent as described by the manufacturer (Life Technologies, Inc., Gaithersburg, MD), Dnase I treated (Promega Corporation, Madison, WI) and purified using an RNeasy spin-column (Qiagen, Valencia, CA).  The quality and balance of the RNA samples were tested by electrophoresis on a denaturing agarose gel.

Hybridization and analysis of the Affymetrix GeneChip Murine 11K Set.  The murine 11K set consists of 2 probe arrays (subarray A and B) that allow monitoring of the relative abundance of greater than 11,000 genes selected from the UniGene (8/96 and Build 4.0) and TIGR (Build 1.0 beta) databases (Affymetrix Inc, Santa Clara, CA).  Biotinylated cRNAs were prepared from the pooled RNA samples and fragmented following the manufacturer’s protocol (Affymetrix Inc.).  The murine 11K A and B arrays were prehybridized and hybridized as instructed by the manufacturer using the GeneChip Fluidics Station 400 (Affymetrix Inc.).  The probed arrays (n=1) were scanned with a Hewlett Packard Gene Array scanner.  The scanned images were analyzed to generate the quantitations based on the images using GeneChip 3.1 software (Affymetrix Inc.).  The default settings of the software were used for the analysis.  Below are the algorithm parameters for each array.

Algorithm Parameters:	GEO WT_11KSubA
BF=	SDT=294.5(4.00Q)	SRT=1.50	ABS={{3.0,4.0},{0.33,0.43},{0.90,1.30}}	RawQ=20.35
QMult=2.80	RL=10.0	HZ=4	VZ=4	BG=2
CT=294.5	PCT=80	DD={{3.0,4.0},{0.33,0.43},{0.20,0.30},{0.90,1.30}}	AvgNCT=200	FCMin=1.00
FCMax=1.00	STP=3.0	TGT=2500	NF=1.000000	SF=3.618839
SFGene=All

Experiment:	WT-subA
Corner+		Avg:1618		Count:32
Corner-		Avg:45546		Count:32
Central-		Avg:45546		Count:9
Edge+		Avg:1493		Count:8
Edge-		Avg:45546		Count:8
Background	Avg: 626	Stdv: 19.7	Min: 589	Max: 662
Pixels	Avg: 36	Stdv: 1.2	Min: 25	Max: 36
Keywords = CEBPE peritoneal macrophage neutrophil knockout

source_name: Total RNA from peritoneal cells harvested by lavage 24 h post thioglycollate injection


title: sed vs MR (M6-63)

description:
Source1 (Channel 1; Cy5)= Middle Runners (6 male C57Bl/6J mice); pooled RNA from the hippocampus of 6 MR (ran 2-5 km/night).

Source2 (Channel 2; Cy3)= Sedentary controls (6 male C5lBl/6J mice); pooled RNA from the hippocampus of 6 sed (no running).

Total RNA was extracted from the frozen hippocampi of individual animals using TRI REAGENT® (Molecular Research Center, Inc, Cincinnati, OH), followed by purification on Qiagen columns from the RNAeasy kit. Equal amounts (6-mg) of total RNA from each of the randomly chosen middle (n=6) MR and sedentary controls (n=6) were combined to generate 2 separate pools. Pooled total RNA was primed with oligo(dT) for reverse transcription, labeled with Cy3 and Cy5 dyes using the Micromax TSA Labeling and Detection Kit protocol (Perkin Elmer Life Sciences, Inc., Boston, MA) and hybridized to the in-house spotted slides. Six microarrays were hybridized (3 forward-labeled, 3 reverse-labeled, to account for differential dye incorporation) per comparison. The protocol for labeling and hybridization was basically as follows. The differentially fluorescent-labeled first-strand cDNA from each source was suspended in 22 µl of hybridization solution containing 5µl of poly-A (5mg/ml), 12.5µl of 20x SSC, 0.5µl 10% SDS and 12.5µl deionized formamide, combined and hybridized to the arrays for 30-36 hrs. The signals from specifically hybridized biotin- and fluorescein-labeled cDNAs were amplified either with streptavidin-horseradish peroxidase (HRP) and Cy5-tyramide or antifluorescein-HRP and Cy3-tyramide, respectively. Fluorescent array images were collected with a GSI Lumonics ScanArray 5000 fluorescent scanner (Packard Biochip Technologies; Meriden, CT) and image intensity data were extracted and analyzed using the provided QuantArray V. 3.0 software (Packard Bioscience; Meriden, CT). A uniform scale factor was applied to normalize signal intensities between Cy5 and Cy3. LOWESS normalization was applied to account for dye variation across and between slides, and for signal intensity effects. An in-house software program was used to perform this analysis (Details of the algorithm can be found at http://bioinfo.pbrc.edu). Fluorescent intensities below 500 were removed, and the expression ratios were LOWESS transformed to obtain the log base 2 values given in the data table.

Keywords = hippocampus
Keywords = voluntary running
Keywords = C57Bl/6J
Lot batch = PBRC M6-63


title: det2_Mock2

source_name: Genotype: Columbia-0, det2 mut.; Treatment: incubated with 80 percent ethanol for 3 hours; Biological Replicate: 2 of three


title: Wildtype 3 (jos0928)

status: Public on Jun 01 2005

city: Boston

first: Atul

last: Butte

state: MA

address: 300 Longwood Avenue

institute: Children's Hospital, Boston

description:
Brown preadipocyte cell lines derived from newborn wildtype littermates of IRS-1 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation. Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described. 15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA). Intensity values were quantitated by MAS 5.0 software (Affymetrix). All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 928

source_name: Mouse brown preadipocyte cell line


title: bzr1-1D_Mock3

description:
Growth Conditions and Treatments: Arabidopsis thaliana seeds were surface sterilized. Approximately 15000 seeds(300 milligrams) were placed in a 1 L erlenmeyer flasks containing 300 mL of liquid Murashige and Skoog medium (Sigma, St Louis, MO, USA) containing 1.5% sucrose. These flasks were incubated with gentle shaking for 5 days under constant illumination at 22ºC. After this time, 15 ul of 80% ethanol was added as the mock control (BL treated received 15 ul of 2uM Brassinolide (BL) dissolved in 80% ethanol.  The BL treatment results were not used and are thus not reported). Flasks were incubated for 3 hours with the ethanol. Whole seedlings from each treatment were collected, the medium was drained, and the seedlings were rapidly forzen in liquid nitrogen. The experiment was done alongside two other genetic backgrounds (wildtype and det2) and was repeated three times (experiments 1-3) during different weeks.
RNA and Microarray Methods: Total RNA was extracted from the plants using a modified Trizol method (Chomczynski and Sashi, 1987)(see also Protocols Manual at the AFGC web page http://www.arabidopsis.org/info/2010_projects/comp_proj/AFGC/RevisedAFGC/AFGC_Protocols_Dec_2001L.pdf) and purified with a silica membrane column (Qiagen, RNeasy). Fifteen micrograms biotinylated complementary RNA (cRNA) was prepared and used to hybridize ATH1 Arabidopsis GeneChips (Affymetrix) using the manufacturer’s protocols. The array images were analyzed with the Affymetrix GeneChip Operating Software (GCOS) 1.1 with the target intensity set to 500. These data were imported into GeneSpring 7.0. To remove chip-to-chip and experiment-to-experiment signal variation each array was normalized by the intensity values of the top 50th percentile of genes on this array. The individual detection values were then normalized to the corresponding detection values of the wildtype (Colombia-0) control for each experiment. The normalized values for the data set are reported along with the intensity values for this array.
Keywords = Arabidopsis, hormone, brassinosteroid, det2, bzr1-1D
Lot batch = 510690

source_name: Genotype: Columbia-0, bzr1-1D mut.; Treatment: incubated with 80 percent ethanol for 3 hours; Biological Replicate: 3 of three


title: det2_Mock1

source_name: Genotype: Columbia-0, det2 mut.; Treatment: incubated with 80 percent ethanol for 3 hours; Biological Replicate: 1 of three


title: bzr1-1D_Mock2

source_name: Genotype: Columbia-0, bzr1-1D mut.; Treatment: incubated with 80 percent ethanol for 3 hours; Biological Replicate: 2 of three


title: Wildtype 1 (jos0532)

description:
Brown preadipocyte cell lines derived from newborn wildtype littermates of IRS-1 KO mice. Preadipocytes were grown to confluence (day 0) in differentiation medium (Dulbeco’s modified Earle’s medium containing 10% fetal bovine serum supplemented with 20 nM insulin and 1 nM T3). Brown preadipocytes were synchronized by overnight serum starvation.  Total RNA was isolated with ULTRASPEC RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX) following the manufacture’s instruction. 28 ug of total RNA were subjected to cDNA and cRNA preparations as previously described.  15 ug of adjusted cRNA were hybridised to Affymetrix U74A-v2 arrays (Santa Clara, CA).  Intensity values were quantitated by MAS 5.0 software (Affymetrix).  All chips were subjected to global scaling to a target intensity of 1,500 to take into account the inherent differences between the chips and their hybridization efficiencies.
Keywords = brown fat
Keywords = preadipocyte
Keywords = mouse
Lot batch = Joslin core 532


title: BM4_RLO_U133A

status: Public on May 19 2005

city: Minneapolis

first: Craig

middle: E

last: Eckfeldt

state: MN

address: 420 Delaware St. SE, MMC 806

department: Stem Cell Institute

institute: University of Minnesota

description: Adult human BM was obtained after informed consent in accordance with guidelines approved by the University of Minnesota Committee on the Use of Human Subjects in Research. Each biologically-distinct replicate was comprised of a single donor. CD34+CD38-CD33-Rho(lo)c-kit+ and CD34+CD38-CD33-Rho(hi) fractions were selected by sequential Ficol-Hypaque separation, MACS column depletion and fluorescence activated cell sorting. Total cellular RNA was isolated using the PicoPure RNA Isolation Kit (Arcturus, Mountain View, California) per the manufacturer’s instructions. Seven to ten thousand Rho(lo) and Rho(hi) cells were sorted directly into 100 mcL Extraction Buffer (XB) provided with the PicoPure RNA Isolation Kit (Arctutus) prior to RNA isolation. Labeled complimentary-RNA (cRNA) was generated by one round of IVT-based, linear amplification using the RiboAmp OA RNA Amplification Kit (Arcturus) followed by labeling with the Enzo Bioarray™ HighYield™ RNA Transcript Labeling Kit (Enzo Life Sciences, Farmingdale, New York) according to manufacturer’s instructions. Affymetrix® HG-U133 GeneChips™ were processed using Genedata Refiner software (GeneData, San Francisco, California) to assess overall quality. Feature intensities for each chip were condensed into a single intensity value per gene using the Affymetrix Statistical Algorithm (MAS 5.0) with Tau = 0.015, Alpha1 = 0.04, Alpha2 0.06 and a scaling factor of 500.

organism: Homo sapiens

source_name: bone marrow CD34+CD38-CD33-Rho(lo)c-kit+ cells


title: BM3_RLO_U133A


title: BM2_RHI_U133A

source_name: bone marrow CD34+CD38-CD33-Rho(hi) cells


title: CB5_RLO_U133A

description: Human UCB from full-term delivered infants was obtained after informed consent in accordance with guidelines approved by the University of Minnesota Committee on the Use of Human Subjects in Research. Each biologically-distinct replicate was comprised of one to four donors that were pooled prior to processing and microarray analysis. CD34+CD38-CD33-Rho(lo)c-kit+ and CD34+CD38-CD33-Rho(hi) fractions were selected by sequential Ficol-Hypaque separation, MACS column depletion and fluorescence activated cell sorting. Total cellular RNA was isolated using the PicoPure RNA Isolation Kit (Arcturus, Mountain View, California) per the manufacturer’s instructions. Seven to ten thousand Rho(lo) and Rho(hi) cells were sorted directly into 100 mcL Extraction Buffer (XB) provided with the PicoPure RNA Isolation Kit (Arctutus) prior to RNA isolation. Labeled complimentary-RNA (cRNA) was generated by one round of IVT-based, linear amplification using the RiboAmp OA RNA Amplification Kit (Arcturus) followed by labeling with the Enzo Bioarray™ HighYield™ RNA Transcript Labeling Kit (Enzo Life Sciences, Farmingdale, New York) according to manufacturer’s instructions. Affymetrix® HG-U133 GeneChips™ were processed using Genedata Refiner software (GeneData, San Francisco, California) to assess overall quality. Feature intensities for each chip were condensed into a single intensity value per gene using the Affymetrix Statistical Algorithm (MAS 5.0) with Tau = 0.015, Alpha1 = 0.04, Alpha2 0.06 and a scaling factor of 500.

source_name: umbilical cord blood CD34+CD38-CD33-Rho(lo)c-kit+ cells


title: BM3_RHI_U133A


title: BM1_RHI_U133A


title: CB3_RHI_U133A

source_name: umbilical cord blood CD34+CD38-CD33-Rho(hi) cells


title: BM1_RLO_U133A


title: bzr1-1D_Mock1

source_name: Genotype: Columbia-0, bzr1-1D mut.; Treatment: incubated with 80 percent ethanol for 3 hours; Biological Replicate: 1 of three


title: CB3_RLO_U133A


title: CB1_RHI_U133A


title: CB4_RHI_U133A


title: CB4_RLO_U133A


title: BM2_RLO_U133A


title: BM4_RHI_U133B


title: CB1_RLO_U133A


title: CB5_RHI_U133A


title: BM3_RHI_U133B


title: BM3_RLO_U133B


title: BM4_RLO_U133B


title: BM4_RHI_U133A


title: CB2_RLO_U133A


title: CB2_RHI_U133A


title: BM2_RLO_U133B


title: BM1_RLO_U133B


title: CB5_RLO_U133B


title: Female J90 - 1

city: Rennes

first: Daniel

last: Baron

state: Brittany

department: Station Commune de Recherches en Ichtyophysiologie

country: France

institute: Institut National de la Recherche Agronomique-INRA

description:
Large-scale temporal gene expression profiling during gonadal differentiation and early gametogenesis in rainbow-trout
Animals and samplings:
Research involving animal experimentation has been approved by the author's institution (authorization number 35-14) and conforms to NIH guidelines. All-male and all-female rainbow trout populations were obtained from the INRA experimental fish farm (Drennec, France) using breeders from the Mirwart strain. At 55 days post-fertilization (55 dpf, i.e., the onset of the free swimming), a male and female batch of 1500 fry each were transferred in 0.3 m3 tanks with recirculating water conditions. They were held at 12°C under constant photoperiod (12h light : 12h dark) and fed ad libitum with a commercial diet (dry pellet food, BiomarTM, Brande, Denmark). In each group, from 20 to 100 gonads, depending on the age of the fish, were sampled and pooled in duplicates at various stages of development : the onset of the free swimming period after complete yolk resumption (Day 0 = D0), D7, D12 (around the first occurrence of oocyte meiosis), D27 (first ovarian lamellar structures), D60 (first previtellogenic oocytes), D90 and D110. There were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Additional gonads were sampled at the same dates for histological analysis performed as previously described.
Total RNA extraction and Reverse Transcription:
Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). Total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip kit (Agilent Technologies, Stockport, UK) according to the manufacturer's instructions. For cDNA synthesis, 1µg of RNA was denaturated in the presence of random hexamers (0.5µg) for 5 min at 70°C, and then chilled on ice. Reverse transcription (RT) was performed at 37°C for 1 h using M-MLV reverse transcriptase (Promega, Madison, WI) as described by the manufacturer.
Primers design:
Trout candidate gene homologues were searched in international public databanks using a reciprocal blast hit strategy. Candidate genes were chosen according to a bibliographic analysis showing their direct or indirect involvement in the sex differentiation cascade in vertebrates. All the primers were purchased from Eurogentec (Eurogentec, Seraing, Belgium). These primers were manually designed respecting whenever possible the following restriction parameters : 21-23 bp length, no more than 4 identical successive nucleotides, 30-70% GC content, a maximum of 2 G or C among the five 3'-end bases, no primer dimers and a short amplicon size (70-150 bp). Secondary structures were searched with DNA mfold (http://bioinfo.math.rpi.edu/~mfold/DNA/form1.cgi). Whenever possible, each pair was chosen with at least one primer flanking an intron-exon boundary, in order to prevent genomic amplification.
Real-time RT-PCR
Real-time RT-PCR was carried out on an iCycler iQTM (BioRad, Hercules, CA). Reactions were performed in 20µl with 300nM of each primer, 5µl of a 1/50 dilution of the RT reaction and the SYBER-Green PCR master Mix (Eurogentec) according to the manufacturer's instructions. After two incubation steps (50°C 10 min, 95°C 2 min), the thermal cycling protocol was 95°C for 10 min followed by 40 cycles of PCR (95°C 30 s, 60°C or 65°C 1 min). For each primer set the efficiency of the PCR reaction (linear equation : y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse transcribed RNA samples). Real-time PCR efficiencies for each reaction were then calculated using the formula : Efficiency (E)=[10(1/slope)]-1. Melting curve analysis was also performed for each gene to check the specificity and identity of the RT-PCR products. The relative amount of the target RNA called the Starting Quantity (SQ) was then determined using the I-Cycler IQ software by comparison with the corresponding standard curve for each sample run in duplicate. SQ were calculated as follow : SQ = [10((Ct -intercept)/slope))]-1 where Ct is the Cycle threshold of the unknown sample. Each transcript level was then normalized by division with the expression values of the constitutive elongation factor 1 alpha (EF1a) used as an internal standard.

organism: Oncorhynchus mykiss

source_name: Female J90 - 1


title: CB2_RHI_U133B


title: CB3_RHI_U133B


title: CB2_RLO_U133B


title: CB5_RHI_U133B


title: CB4_RHI_U133B


title: CB1_RLO_U133B


title: CB3_RLO_U133B


title: BM1_RHI_U133B


title: WT_P_1

status: Public on May 14 2005

first: Zhe

last: Li

department: Division of Hematology/Oncology

institute: Children's Hospital Boston

description:
E12.5 fetal liver (FL) cells from wild type embryos were cultured ex vivo in the presence of Tpo for three days. FL-derived megakaryocytes and their progenitors in the culture were separated by BSA density gradient. Megakaryocyte progenitors (“P” population) were further purified by fluorescence-activated cell sorting (FACS) for cells expressing low levels of CD41. Megakaryocytes (“M” population) were further enriched by CD41 antibody coupled with magnetic beads. Total RNA was isolated from either enriched megakaryocyte or progenitor fractions. ~5-15microgram RNA from each sample was submitted to the Dana Farber Cancer Institute Microarray Core Facility for cDNA and cRNA synthesis, labeling, and hybridization with Affymetrix Mouse Genome 430 2.0 chips according to manufacturer’s instructions. All arrays were normalized and analyzed using the dChip program.
Keywords = GATA-1
Keywords = megakaryocyte
Keywords = mouse model

source_name: wild type E12.5 fetal liver derived megakaryocyte progenitors


title: Low_M_1

description:
E12.5 fetal liver (FL) cells from GATA-1deltaNeodeltaHS mutant  embryos were cultured ex vivo in the presence of Tpo for three days. FL-derived megakaryocytes and their progenitors in the culture were separated by BSA density gradient. Megakaryocyte progenitors (“P” population) were further purified by fluorescence-activated cell sorting (FACS) for cells expressing low levels of CD41. Megakaryocytes (“M” population) were further enriched by CD41 antibody coupled with magnetic beads. Total RNA was isolated from either enriched megakaryocyte or progenitor fractions. ~5-15microgram RNA from each sample was submitted to the Dana Farber Cancer Institute Microarray Core Facility for cDNA and cRNA synthesis, labeling, and hybridization with Affymetrix Mouse Genome 430 2.0 chips according to manufacturer’s instructions. All arrays were normalized and analyzed using the dChip program.
Keywords = GATA-1
Keywords = megakaryocyte
Keywords = mouse model

source_name: GATA-1deltaNeodeltaHS mutant E12.5 fetal liver derived megakaryocytes


title: CB1_RHI_U133B


title: Low_M_2


title: left ventricular myocardium, non-failing 5B

status: Public on May 13 2005

city: Munich

first: Andreas

middle: S.

last: Barth

address: Marchioninistr. 15

department: Department of Cardiology

country: Germany

institute: University of Munich (LMU), Grosshadern

description:
Right atrial appendages were obtained from 30 patients undergoing open heart surgery for valve repair or coronary artery bypass grafting. Of these, ten patients had permanent AF defined as duration of AF longer than three months as documented by ECG, and 20 patients had no history of AF and were in sinus rhythm when open heart surgery was performed. Transmural LV tissue was obtained from five non-failing donor hearts which were not transplanted due to palpable coronary calcifications. After excision, all tissue specimens were immediately frozen in liquid nitrogen and stored at -80°C. Total RNA was purified from homogenized deep-frozen tissue samples following the TRIZOL standard protocol as described by the manufacturer (GibcoBRL, Eggenstein, Germany) and quantified by photometry. 150ng total RNA was used for quality control using the RNA 6000 Nano LabChip kit and Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). Synthesis of double-stranded cDNA and of biotinylated cRNA probes as well as subsequent steps of hybridization, washing, and staining followed instructions provided by Affymetrix (Affymetrix, Santa Clara, CA, USA). One set of U133A+B chips (together representing 44.928 probe sets) was used for each human heart sample. All U133 GeneChips passed quality control to eliminate scans with abnormal characteristics, i.e. abnormal low or high dynamic range, high perfect match saturation, high pixel noise, grid misalignment problems, and low mean signal to noise ratio. Low level and statistical analysis was done using R 1.8.1. Microarray data of all atrial and ventricular samples was normalized simultaneously using robust multi-array average (RMA) implemented in Bioconductor's "affy"-package.
Keywords = Homo sapiens, myocardial tissue, comparison of right atrial tissue of patients with atrial fibrillation and sinus rhythm with left ventricular gene expression

source_name: myocardium, left ventricular free wall


title: Mut_M_2

description:
E12.5 fetal liver (FL) cells from GATA-1deltaN mutant embryos were cultured ex vivo in the presence of Tpo for three days. FL-derived megakaryocytes and their progenitors in the culture were separated by BSA density gradient. Megakaryocyte progenitors (“P” population) were further purified by fluorescence-activated cell sorting (FACS) for cells expressing low levels of CD41. Megakaryocytes (“M” population) were further enriched by CD41 antibody coupled with magnetic beads. Total RNA was isolated from either enriched megakaryocyte or progenitor fractions. ~5-15microgram RNA from each sample was submitted to the Dana Farber Cancer Institute Microarray Core Facility for cDNA and cRNA synthesis, labeling, and hybridization with Affymetrix Mouse Genome 430 2.0 chips according to manufacturer’s instructions. All arrays were normalized and analyzed using the dChip program.
Keywords = GATA-1
Keywords = megakaryocyte
Keywords = mouse model

source_name: GATA-1deltaN mutant E12.5 fetal liver derived megakaryocytes


title: WT_P_2


title: Mut_P_1

description:
E12.5 fetal liver (FL) cells from GATA-1deltaN mutant  embryos were cultured ex vivo in the presence of Tpo for three days. FL-derived megakaryocytes and their progenitors in the culture were separated by BSA density gradient. Megakaryocyte progenitors (“P” population) were further purified by fluorescence-activated cell sorting (FACS) for cells expressing low levels of CD41. Megakaryocytes (“M” population) were further enriched by CD41 antibody coupled with magnetic beads. Total RNA was isolated from either enriched megakaryocyte or progenitor fractions. ~5-15microgram RNA from each sample was submitted to the Dana Farber Cancer Institute Microarray Core Facility for cDNA and cRNA synthesis, labeling, and hybridization with Affymetrix Mouse Genome 430 2.0 chips according to manufacturer’s instructions. All arrays were normalized and analyzed using the dChip program.
Keywords = GATA-1
Keywords = megakaryocyte
Keywords = mouse model

source_name: GATA-1deltaN mutant E12.5 fetal liver derived megakaryocyte progenitors


title: WT_M_1

source_name: wild type E12.5 fetal liver derived megakaryocytes


title: CB4_RLO_U133B


title: WT_M_2


title: left ventricular myocardium, non-failing 4B


title: left ventricular myocardium, non-failing 3

description:
Right atrial appendages were obtained from 30 patients undergoing open heart surgery for valve repair or coronary artery bypass grafting. Of these, ten patients had pmAF defined as duration of AF longer than three months as documented by ECG, and 20 patients had no history of AF and were in SR when open heart surgery was performed. Transmural LV tissue was obtained from five non-failing donor hearts which were not transplanted due to palpable coronary calcifications. After excision, all tissue specimens were immediately frozen in liquid nitrogen and stored at -80°C. Total RNA was purified from homogenized deep-frozen tissue samples following the TRIZOL standard protocol as described by the manufacturer (GibcoBRL, Eggenstein, Germany) and quantified by photometry. 150ng total RNA was used for quality control using the RNA 6000 Nano LabChip kit and Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). Synthesis of double-stranded cDNA and of biotinylated cRNA probes as well as subsequent steps of hybridization, washing, and staining followed instructions provided by Affymetrix (Affymetrix, Santa Clara, CA, USA). One set of U133A+B chips (together representing 44.928 probe sets) was used for each human heart sample. All U133 GeneChips passed quality control to eliminate scans with abnormal characteristics, i.e. abnormal low or high dynamic range, high perfect match saturation, high pixel noise, grid misalignment problems, and low mean signal to noise ratio. Low level and statistical analysis was done using R 1.8.1. Microarray data of all atrial and ventricular samples was normalized simultaneously using robust multi-array average (RMA) implemented in Bioconductor's "affy"-package.
Keywords = Homo sapiens, myocardial tissue, comparison of right atrial tissue of patients with atrial fibrillation and sinus rhythm with left ventricular gene expression


title: Mut_M_1


title: Mut_P_2


title: left ventricular myocardium, non-failing 5


title: left ventricular myocardium, non-failing 3B


title: left ventricular myocardium, non-failing 4


title: left ventricular myocardium, non-failing 2B


title: left ventricular myocardium, non-failing 1


title: left ventricular myocardium, non-failing 1B


title: atrial myocardium, sinus rhythm 19B

source_name: myocardium, right atrial appendage


title: atrial myocardium, sinus rhythm 20B


title: atrial myocardium, sinus rhythm 17


title: atrial myocardium, sinus rhythm 19


title: atrial myocardium, sinus rhythm 16


title: atrial myocardium, sinus rhythm 20


title: atrial myocardium, sinus rhythm 15


title: atrial myocardium, sinus rhythm 15B


title: atrial myocardium, sinus rhythm 16B


title: atrial myocardium, sinus rhythm 13


title: atrial myocardium, sinus rhythm 17B


title: atrial myocardium, sinus rhythm 14


title: atrial myocardium, sinus rhythm 18


title: atrial myocardium, sinus rhythm 11B


title: atrial myocardium, sinus rhythm 13B


title: atrial myocardium, sinus rhythm 12


title: atrial myocardium, sinus rhythm 12B


title: atrial myocardium, sinus rhythm 10B


title: atrial myocardium, sinus rhythm 14B


title: atrial myocardium, sinus rhythm 10


title: atrial myocardium, sinus rhythm 11


title: atrial myocardium, sinus rhythm 9B


title: atrial myocardium, sinus rhythm 7B


title: atrial myocardium, sinus rhythm 8B


title: atrial myocardium, sinus rhythm 7


title: BM2_RHI_U133B


title: atrial myocardium, sinus rhythm 8


title: atrial myocardium, sinus rhythm 9


title: atrial myocardium, sinus rhythm 3B


title: atrial myocardium, sinus rhythm 4B


title: left ventricular myocardium, non-failing 2


title: atrial myocardium, sinus rhythm 18B


title: atrial myocardium, sinus rhythm 4


title: atrial myocardium, sinus rhythm 3


title: atrial myocardium, atrial fibrillation 10B


title: atrial myocardium, sinus rhythm 5B


title: atrial myocardium, sinus rhythm 6


title: atrial myocardium, sinus rhythm 1


title: atrial myocardium, sinus rhythm 5


title: atrial myocardium, atrial fibrillation 8B


title: atrial myocardium, sinus rhythm 2B


title: atrial myocardium, atrial fibrillation 9B


title: atrial myocardium, atrial fibrillation 10


title: atrial myocardium, sinus rhythm 2


title: atrial myocardium, atrial fibrillation 9


title: atrial myocardium, sinus rhythm 6B


title: atrial myocardium, atrial fibrillation 8


title: atrial myocardium, sinus rhythm 1B


title: atrial myocardium, atrial fibrillation 7B


title: atrial myocardium, atrial fibrillation 7


title: atrial myocardium, atrial fibrillation 6


title: atrial myocardium, atrial fibrillation 4


title: atrial myocardium, atrial fibrillation 6B


title: atrial myocardium, atrial fibrillation 4B


title: atrial myocardium, atrial fibrillation 5B


title: atrial myocardium, atrial fibrillation 5


title: atrial myocardium, atrial fibrillation 3B


title: PFR3_replicate_4

status: Public on May 06 2005

city: Irvine

first: Barbara

last: Apostol

department: Psychiratry and Human Behavior

institute: University of California Irvine

description: For gene expression profiling studies three clones of each line (see above), PFR3 or PFR3K650E cells, were plated in 150-mm plates and the following day induced for 24 hr.  Total RNA was isolated separately for each clone using Trizol Reagent according to the manufacturer (Invitrogen).  RNA was quantitated spectrophotometrically and integrity tested by capillary electrophoresis (Agilent 2100 Bioanalyzer).  Equal amounts of RNA from each clone of mutant and wild type lines were pooled and a total of 25 µg total RNA was used to generate target cRNAs for hybridization to Affymetrix Rat Genome U34A oligonculeotide arrays (UCI DNA Array Core Facility).  Three separate cell growths were done for each clone and cRNAs were synthesized from the pooled RNAs separately and triplicate GeneChips were done.  Gene expression profiling is described in Supplemental Text.

organism: Rattus norvegicus

source_name: PC12 line PFR3


title: atrial myocardium, atrial fibrillation 2


title: PFR3_replicate_3


title: atrial myocardium, atrial fibrillation 2B


title: atrial myocardium, atrial fibrillation 1B


title: atrial myocardium, atrial fibrillation 3


title: minipump male bGH 1 week (1.4)

status: Public on May 04 2005

city: Stockholm

first: Amilcar

last: Flores

country: Sweden

institute: Karolinska Institute

description: Young male Sprague Dawley rats. 4 control animals were treated with vehicle and 4 animals were treated with recombinant bovine growth hormone (bGH) by continous infusion from osmotic minipumps for 1 week before they were killed. The bGH dose was 5 µg/hour.

source_name: Untreated

source_name: rat, liver, growth hormone, minipump male bGH 1 week Cy3 Total RNA


title: atrial myocardium, atrial fibrillation 1

description:
Right atrial appendages were obtained from 30 patients undergoing open heart surgery for valve repair or coronary artery bypass grafting. Of these, ten patients had pmAF defined as duration of AF longer than three months as documented by ECG, and 20 patients had no history of AF and were in SR when open heart surgery was performed. Transmural LV tissue was obtained from five non-failing donor hearts which were not transplanted due to palpable coronary calcifications. After excision, all tissue specimens were immediately frozen in liquid nitrogen and stored at -80°C. Total RNA was purified from homogenized deep-frozen tissue samples following the TRIZOL standard protocol as described by the manufacturer (GibcoBRL, Eggenstein, Germany) and quantified by photometry. 150ng total RNA was used for quality control using the RNA 6000 Nano LabChip kit and Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). Synthesis of double-stranded cDNA and of biotinylated cRNA probes as well as subsequent steps of hybridization, washing, and staining followed instructions provided by Affymetrix (Affymetrix, Santa Clara, CA, USA). One set of U133A+B chips (together representing 44.928 probe sets) was used for each human heart sample. All U133 GeneChips passed quality control to eliminate scans with abnormal characteristics, i.e. abnormal low or high dynamic range, high perfect match saturation, high pixel noise, grid misalignment problems, and low mean signal to noise ratio. Low level and statistical analysis was done using R 1.8.1. Microarray data of all atrial and ventricular samples was normalized simultaneously using robust multi-array average (RMA) implemented in Bioconductor's "affy"-package.
Keywords = Homo sapiens, myocardial tissue, comparison of right atrial tissue of patients of patients with atrial fibrillation and sinus rhythm with left ventricular gene expression


title: PFR3_replicate_1


title: PFR3K650E_replicate_4

source_name: PC12 line PFR3K650E


title: PFR3K650E_replicate_3


title: minipump male bGH 1 week (1.2)

source_name: rat, liver, growth hormone, minipump male bGH 1 week Cy5 Total RNA


title: minipump male bGH 1 week (1.3)


title: minipump male bGH 1 week (1.1)

source_name: rat, liver, growth hormone, minipump male bGH 1 week  Cy5 Total RNA


title: PFR3K650E_replicate_2


title: spike_in_concentration_0.75pmol(self-hybridisation)_replicate_5

status: Public on May 03 2005

city: Singappore

first: Althea

last: Chua

country: Singapore

institute: Bioinformatics Institute

description:
Obtained from the American Type Culture Collection.
Fusion of antigen-primed BALB/c mouse spleen cells and the BALB/c mouse myeloma P3-X63-Ag8
produces a monoclonal antibody against human fibronectin.
CRL1606 was cultivated as suspension cultures in shake flasks placed in humidified incubators (95% relative humidity) at 37?C, 8% CO2
The cells were routinely maintained in a serum-free media formulation based on Iscove’s modified Dulbecco’s medium (IMDM) and passaged every 2 to 2.5 days, inoculating at 3 x 105 cells ml
Protocol for mRNA sample preparation, labelling, hybridization, scanning and normalization can be found on the web-link. Normalized data can also be found on the web-link.

source_name: mouse hybridoma, ATCC CRL1606 plus 100 genes spiked at a concentration of 0.75pmol for each gene

source_name: mouse hybridoma, ATCC CRL1606 plus another 100 genes spiked at a concentration of 0.75pmol for each gene


title: spike_in_concentration_0.75pmol(self-hybridisation)_replicate_4


title: spike_in_concentration_2pmol(self-hybridisation)_replicate_2

source_name: mouse hybridoma, ATCC CRL1606 plus 100 genes spiked at a concentration of 2pmol for each gene

source_name: mouse hybridoma, ATCC CRL1606 plus another 100 genes spiked at a concentration of 2pmol for each gene


title: PFR3_replicate_2


title: spike_in_concentration_1pmol(self-hybridisation)_replicate_3

source_name: mouse hybridoma, ATCC CRL1606 plus 100 genes spiked at a concentration of 1pmol for each gene

source_name: mouse hybridoma, ATCC CRL1606 plus another 100 genes spiked at a concentration of 1pmol for each gene


title: spike_in_concentration_0.75pmol(self-hybridisation)_replicate_6


title: PFR3K650E_replicate_1


title: spike_in_concentration_2pmol(self-hybridisation)_replicate_3


title: spike_in_concentration_2pmol(self-hybridisation)_replicate_1


title: spike_in_concentration_0.75pmol(self-hybridisation)_replicate_3


title: spike_in_concentration_1pmol(self-hybridisation)_replicate_1


title: spike_in_concentration_0.5pmol(self-hybridisation)_replicate_4

source_name: mouse hybridoma, ATCC CRL1606 plus 100 genes spiked at a concentration of 0.5pmol for each gene

source_name: mouse hybridoma, ATCC CRL1606 plus another 100 genes spiked at a concentration of 0.5pmol for each gene


title: spike_in_concentration_0.75pmol(self-hybridisation)_replicate_2


title: spike_in_concentration_1pmol(self-hybridisation)_replicate_2


title: spike_in_concentration_0.5pmol(self-hybridisation)_replicate_2


title: spike_in_concentration_0.75pmol(self-hybridisation)_replicate_1


title: spike_in_concentration_0.5pmol(self-hybridisation)_replicate_5


title: spike_in_concentration_0.25pmol(self-hybridisation)_replicate_4

source_name: mouse hybridoma, ATCC CRL1606 plus 100 genes spiked at a concentration of 0.25pmol for each gene

source_name: mouse hybridoma, ATCC CRL1606 plus another 100 genes spiked at a concentration of 0.25pmol for each gene


title: spike_in_concentration_0.5pmol(self-hybridisation)_replicate_6


title: spike_in_concentration_0.25pmol(self-hybridisation)_replicate_6


title: spike_in_concentration_0.25pmol(self-hybridisation)_replicate_3


title: spike_in_concentration_0.25pmol(self-hybridisation)_replicate_5


title: spike_in_concentration_0.5pmol(self-hybridisation)_replicate_1


title: spike_in_concentration_0.2pmol(self-hybridisation)_replicate_3

source_name: mouse hybridoma, ATCC CRL1606 plus 100 genes spiked at a concentration of 0.2pmol for each gene

source_name: mouse hybridoma, ATCC CRL1606 plus another 100 genes spiked at a concentration of 0.2pmol for each gene


title: spike_in_concentration_0.5pmol(self-hybridisation)_replicate_3


title: spike_in_concentration_0.25pmol(self-hybridisation)_replicate_1


title: spike_in_concentration_0.25pmol(self-hybridisation)_replicate_2


title: spike_in_concentration_0.2pmol(self-hybridisation)_replicate_2


title: spike_in_concentration_0.15pmol(self-hybridisation)_replicate_3

source_name: mouse hybridoma, ATCC CRL1606 plus 100 genes spiked at a concentration of 0.15pmol for each gene

source_name: mouse hybridoma, ATCC CRL1606 plus another 100 genes spiked at a concentration of 0.15pmol for each gene


title: spike_in_concentration_0.05pmol(self-hybridisation)_replicate_4

source_name: mouse hybridoma, ATCC CRL1606 plus 100 genes spiked at a concentration of 0.05pmol for each gene

source_name: mouse hybridoma, ATCC CRL1606 plus another 100 genes spiked at a concentration of 0.05pmol for each gene


title: spike_in_concentration_0.1pmol(self-hybridisation)_replicate_2

source_name: mouse hybridoma, ATCC CRL1606 plus 100 genes spiked at a concentration of 0.1pmol for each gene

source_name: mouse hybridoma, ATCC CRL1606 plus another 100 genes spiked at a concentration of 0.1pmol for each gene


title: spike_in_concentration_0.1pmol(self-hybridisation)_replicate_1


title: spike_in_concentration_0.05pmol(self-hybridisation)_replicate_6


title: spike_in_concentration_0.025pmol(self-hybridisation)_replicate_2

source_name: mouse hybridoma, ATCC CRL1606 plus 100 genes spiked at a concentration of 0.025pmol for each gene

source_name: mouse hybridoma, ATCC CRL1606 plus another 100 genes spiked at a concentration of 0.025pmol for each gene


title: spike_in_concentration_0.05pmol(self-hybridisation)_replicate_5


title: spike_in_concentration_0pmol(self-hybridisation)_replicate_2

source_name: mouse hybridoma, ATCC CRL1606


title: spike_in_concentration_0.2pmol(self-hybridisation)_replicate_1


title: spike_in_concentration_0.05pmol(self-hybridisation)_replicate_3


title: spike_in_concentration_0.1pmol(self-hybridisation)_replicate_4


title: spike_in_concentration_0pmol(self-hybridisation)_replicate_3


title: spike_in_concentration_0.05pmol(self-hybridisation)_replicate_2


title: spike_in_concentration_0.025pmol(self-hybridisation)_replicate_1


title: spike_in_concentration_0pmol(self-hybridisation)_replicate_5


title: spike_in_concentration_0.1pmol(self-hybridisation)_replicate_6


title: spike_in_concentration_0pmol(self-hybridisation)_replicate_4


title: spike_in_concentration_0.1pmol(self-hybridisation)_replicate_3


title: spike_in_concentration_0pmol(self-hybridisation)_replicate_6


title: spike_in_concentration_0.1pmol(self-hybridisation)_replicate_5


title: spike_in_concentration_0.15pmol(self-hybridisation)_replicate_1


title: spike_in_concentration_0.05pmol(self-hybridisation)_replicate_1


title: spike_in_concentration_0.025pmol(self-hybridisation)_replicate_3


title: 293T cells treated with 2µM camptothecin for 48h (sample 2)

status: Public on May 02 2005

city: La Jolla

first: Bettina

last: Groschel

address: 10010 North Torrey Pines Road

department: Infectious Disease

institute: The Salk Institute

description: Total RNA from 293T cells treated with 2µM camptothecin for 48h (2 samples)was isolated using RNeasy Midi Kit (QIAGEN, Valencia) followed by gene expression profiling on Affymetrix human U133A arrays and analysis by MAS 5.1.

source_name: 293T cells


title: spike_in_concentration_0.15pmol(self-hybridisation)_replicate_2


title: 9 (Cy3) - 8 (Cy5)

status: Public on Apr 28 2005

city: Columbia

first: Raja

middle: S

last: Settivari

state: MO

address: 920 E Campus Drive

department: ASRC

institute: University of Missouri

description: Rats (n=24) were fed ad libitum diets containing either ground endophyte-free (E-) seed or endophyte–infected (E+) seed for five days at thermoneutrality (21°C).  Rats (n=24) with intraperitonial transmitters were used with core temperature (Tc) and general activity measured every 5 minutes during the study. At treatment end, the liver was removed, weighed and frozen in liquid nitrogen. Twelve rats were selected for microarray experiments, based on degree of sensitivity to fescue toxicosis.  Degree of sensitivity was based on changes in relative liver weights, feed intake, average daily gain, and Tc from pretreatment levels. RNA was extracted from liver samples, converted to cDNA, and hybridized with the printed oligonucleotide slides.

source_name: RAT # 8

source_name: RAT # 9


title: 9 (Cy3) - 25 (Cy5)

source_name: RAT # 25


title: 5 (Cy3) - 17 (Cy5)

source_name: RAT # 17

source_name: RAT # 5


title: 29 (Cy3) - 24 (Cy5)

source_name: RAT # 24

source_name: RAT # 29


title: WT_2

city: Paris

first: Alain

last: Jacquier

institute: Institut Pasteur

description: BMA64 wilde type (Mat a). Grown at 30°C to OD 0.6 in YPD.

organism: Saccharomyces cerevisiae

source_name: total RNA


title: rrp6_1

description: BMA64 rrp6 delta::HIS3 (Mat a). Grown at 30°C to OD 0.6 in YPD.


title: 29 (Cy3) - 9 Cy (5)


title: WT_1


title: rrp6_2


title: 6 (Cy3) - 24 (Cy5)

source_name: RAT # 6


title: 25 (Cy3) - 23 (Cy5)

source_name: RAT # 23


title: 5 (Cy3) - 13 (Cy5)

source_name: RAT # 13


title: 25 (Cy3) - 16 (Cy5)

source_name: RAT # 16


title: WT_3

description:
WT BMA64 control strain to be compared to the rrp6trf4 strain. Haploid Mat a.
Strain grown at OD 0.6 at 30°C in YPD.


title: 24 (Cy3) - 25 (Cy5)


title: 24 (Cy3) - 19 (Cy5)

source_name: RAT # 19


title: spike_in_concentration_0pmol(self-hybridisation)_replicate_1


title: 23 (Cy3) - 8 (Cy5)


title: 23 (Cy3) - 5 (Cy5)


title: 19 (Cy3) - 23 (Cy5)


title: 17 (Cy3) - 19 (Cy5)


title: 17 (Cy3) - 29 (Cy5)


title: 19 (Cy3) - 9 (Cy5)

source_name: RAT# 19


title: 293T cells treated with 2µM camptothecin for 48h (sample 1)


title: 16 (Cy3) - 6 (Cy5)


title: 13 (Cy3) - 29 (Cy5)


title: IFN/Vit-KO

status: Public on Apr 21 2005

city: Braunschweig

first: Robert

last: Geffers

address: Inhoffenstr. 7

department: Dep. Molecular Bacteriology

institute: HCI - Helmholtz Centre for Infection Research

description: KO Vdr -/- mice; Bone marrow derived macrophages (BMDMs); Briefly, femora and tibia of the hind legs were flushed with cold DMEM (Invitrogen) containing 10 % heat-inactivated fetal calf serum (Biowest), 200 mM L-glutamine, and penicillin/streptomycin (Invitrogen). This complete medium was supplemented with M-CSF (50 ng/ml) and bone marrow cells were cultured at 37°C in a humidified 5% CO2 atmosphere for 7 days. Thioglycolate-elicited macrophages were obtained 4 days after the intraperitoneal injection of sterile 4 % thioglycolate medium. Cells were harvested by peritoneal lavage with cold PBS, 1% FCS and were cultured in RPMI 1640 (Invitrogen) including 10 % heat-inactivated fetal calf serum, L-glutamine, and penicillin/streptomycin (P/S). Macrophages were plated at 8 x 104/well in 96-well plates and cultured in P/S-free medium in the presence or absence of IFN-gamma, 1alpha,25(OH)2D3, or both for 24-48 hours.

source_name: BMDM, 1alpha,25(OH)2D3 and IFNgamma treated, KO Vdr -/-


title: Cisplatin 500 ug 10-1-02

status: Public on Apr 20 2005

city: Groton

first: Gregory

middle: R.

last: Warnes

state: CT

address: Eastern Point Road

institute: Pfizer Inc., Groton, CT - USA

description: BY4730 cultures were exposed to medium containing dilutions of test agent (Cisplatin (Cis), 600mg/mL; Methylmethane sulfonate (MMS), 0.12% v/v; Bleomycin (Bleo), 0.15U/mL; NaCl, 10mg/mL; ethanol, 15% v/v) for 120 min. Control cultures were either mock-treated with the solvent, DMSO (for comparison to Cis, MMS, Bleo) or maintained in media (for comparison to ethanol and NaCl) for 120 mins. Sample preparation and processing procedures were performed according the Affymetrix Genechipâ Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA). Briefly, total RNA was isolated from thawed cell pellets using a hot phenol protocol described by Schmitt et. al. (1990). Total RNA was converted to poly(A)+ mRNA and isolated using Oligotex mRNA kit. Double stranded cDNA was generated by using T7-(dT) 24 oligo primer (Genset Corp., San Diego, CA) and Superscript Double Stranded cDNA Synthesis Kit (Gibco BRL, Carlsbad, CA). The cDNA was used for in vitro transcription (IVT) with Enzo BioArray RNA Transcript Labeling Kit to incorporate biotinylated ribonucleotides. Biotinylated cRNA (15 mg) was fragmented in 40 mM tris-acetate, pH 8.1; 30 mM MgOAc; 100 mM KOAc. Samples were then hybridized with Affymetrix probe array Yeast Genome S98 at 45°C for 16 hours with constant rotation. Hybridized probe arrays were washed and stained using an Affymetrix GeneChipâ Fluidics Station 400 and protocol EukGE-WS2. The chips were scanned using the Affymetrix GeneArray Scanner.

source_name: Yeast BY4730


title: 13 (Cy3) to 6 (Cy5)

source_name: rat # 6

source_name: rat # 13


title: IFN-KO

source_name: BMDM, IFNgamma treated, KO Vdr -/-


title: IFN/Vit-WT

description: wild type mice; Bone marrow derived macrophages (BMDMs); Briefly, femora and tibia of the hind legs were flushed with cold DMEM (Invitrogen) containing 10 % heat-inactivated fetal calf serum (Biowest), 200 mM L-glutamine, and penicillin/streptomycin (Invitrogen). This complete medium was supplemented with M-CSF (50 ng/ml) and bone marrow cells were cultured at 37°C in a humidified 5% CO2 atmosphere for 7 days. Thioglycolate-elicited macrophages were obtained 4 days after the intraperitoneal injection of sterile 4 % thioglycolate medium. Cells were harvested by peritoneal lavage with cold PBS, 1% FCS and were cultured in RPMI 1640 (Invitrogen) including 10 % heat-inactivated fetal calf serum, L-glutamine, and penicillin/streptomycin (P/S). Macrophages were plated at 8 x 104/well in 96-well plates and cultured in P/S-free medium in the presence or absence of IFN-gamma, 1alpha,25(OH)2D3, or both for 24-48 hours.

source_name: wild type; BMDM; IFNgamma and  1alpha,25(OH)2D3 treated


title: Vit-KO

source_name: BMDM, 1alpha,25(OH)2D3 treated, KO Vdr -/-


title: IFN-WT

source_name: wild type; BMDM; IFNgamma treated


title: Cisplatin 650 ug 10-23-02


title: MMS 0.12% 8-9-02


title: 16 (Cy3) - 5 (Cy5)


title: MMS 0.12% 7-9-02


title: Bleomycin 0.1 U/ml 10-22-02


title: Bleomycin 0.15 U/ml 10-15-02


title: EtOH 15% 3-1-02


title: 0-KO

description: Vdr tm1 Rge knockout mice; Bone marrow derived macrophages (BMDMs); Briefly, femora and tibia of the hind legs were flushed with cold DMEM (Invitrogen) containing 10 % heat-inactivated fetal calf serum (Biowest), 200 mM L-glutamine, and penicillin/streptomycin (Invitrogen). This complete medium was supplemented with M-CSF (50 ng/ml) and bone marrow cells were cultured at 37°C in a humidified 5% CO2 atmosphere for 7 days. Thioglycolate-elicited macrophages were obtained 4 days after the intraperitoneal injection of sterile 4 % thioglycolate medium. Cells were harvested by peritoneal lavage with cold PBS, 1% FCS and were cultured in RPMI 1640 (Invitrogen) including 10 % heat-inactivated fetal calf serum, L-glutamine, and penicillin/streptomycin (P/S).

source_name: BMDM, not treated, KO Vdr -/-


title: MMS 0.14% 7-12-02


title: Bleomycin 0.15 U/ml 10-23-02


title: Vit-WT

description:
wild type mice; Bone marrow derived macrophages (BMDMs); Briefly, femora and tibia of the hind legs were flushed with cold DMEM (Invitrogen) containing 10 % heat-inactivated fetal calf serum (Biowest), 200 mM L-glutamine, and penicillin/streptomycin (Invitrogen). This complete medium was supplemented with M-CSF (50 ng/ml) and bone marrow cells were cultured at 37°C in a humidified 5% CO2 atmosphere for 7 days. Thioglycolate-elicited macrophages were obtained 4 days after the intraperitoneal injection of sterile 4 % thioglycolate medium. Cells were harvested by peritoneal lavage with cold PBS, 1% FCS and were cultured in RPMI 1640 (Invitrogen) including 10 % heat-inactivated fetal calf serum, L-glutamine, and penicillin/streptomycin (P/S).
Macrophages were plated at 8 x 10^4/well in 96-well plates and cultured in P/S-free medium in the presence or absence of IFN-gamma, 1alpha,25(OH)2D3, or both for 24-48 hours

source_name: wild type; BMDM; 1alpha,25(OH)2D3 treated


title: 0-WT

description: wild type mice; Bone marrow derived macrophages (BMDMs); Briefly, femora and tibia of the hind legs were flushed with cold DMEM (Invitrogen) containing 10 % heat-inactivated fetal calf serum (Biowest), 200 mM L-glutamine, and penicillin/streptomycin (Invitrogen). This complete medium was supplemented with M-CSF (50 ng/ml) and bone marrow cells were cultured at 37°C in a humidified 5% CO2 atmosphere for 7 days. Thioglycolate-elicited macrophages were obtained 4 days after the intraperitoneal injection of sterile 4 % thioglycolate medium. Cells were harvested by peritoneal lavage with cold PBS, 1% FCS and were cultured in RPMI 1640 (Invitrogen) including 10 % heat-inactivated fetal calf serum, L-glutamine, and penicillin/streptomycin (P/S).

source_name: wild type; BMDM; not treated


title: EtOH 15% 2-22-00


title: NaCl 10% 10-15-02


title: 8 (Cy3)- 13 (Cy5)

source_name: RAT # 8


title: NaCl 9% 10-18-02


title: Control 7-9-02


title: NaCl 8% 10-22-02


title: Control 8-9-02


title: Control 2-22-02


title: Control 10-23-02


title: Control 10-22-02


title: Control 10-15-02


title: Control 10-18-02


title: Control 4-10-02


title: Control 10-1-02


title: CT22 6 hr night

status: Public on Apr 15 2005

city: RTP

first: Gary

middle: A

last: Boorman

state: NC

department: Division of Intramural Research Environmental Toxicology Program

institute: NIEHS

description:
One µg of total RNA from either an
individual rat or from a pooled sample was amplified and labeled with a
fluorescent dye (either Cy3 or Cy5) using the Low RNA Input Linear Amplification
Labeling kit (Agilent Technologies, Palo Alto, CA) following the manufacturer’s
protocol. The amount and quality of the resulting fluorescently labeled cRNA
was assessed using a Nanodrop ND-100 spectrophometer and an Agilent
Bioanalyzer. Equal amounts of Cy3- or Cy5-labeled cRNA were hybridized to the
Agilent Rat Oligo Microarray (Agilent Technologies, Inc., Palo Alto, CA) for 17
hrs, prior to washing and scanning. Data was extracted from the resulting
images using Agilent’s Feature Extraction Software (Agilent Technologies, Inc.,
Palo Alto, CA). For day/night comparisons, two types of complementing
hybridizations were performed: hepatic RNA from individual day rats was
hybridized against a pool made up of RNA from 12 hour offset night rats, and
RNA from individual night rats was hybridized against a pool made up of RNA
from 12-hour offset day rats. Specifically, hepatic RNA samples from three
individual day rats collected ten hours after light on (CT10) were hybridized
against a pooled RNA sample composed of equal aliquots of RNA from the livers
of six night rats collected ten hrs after lights off (CT22); and RNA samples from
Page 8
three individual night rats collected at CT22 were hybridized against a pooled
RNA sample composed of equal aliquots of RNA from the livers of six day rats
collected at CT10. This was replicated in a second study for a total of 24
hybridizations of 12 hour offset samples.
For time of day comparisons, equal aliquots of RNA from the livers of six
rats collected at each of four different times of the circadian day (CT4, CT10, C16
and CT22) were pooled. The two replicate studies thus resulted in 8 pools (two
replicate pools/time point times four time points); each pool was hybridized
against a Universal Rat Reference RNA Standard (Stratagene, La Jolla, CA).

source_name: CT22  6hr night

source_name: Stratagene Rat reference


title: CT4 24 hr light

source_name: CT4  24hr light

source_name: STratagene Reference Rat


title: Control 3-1-02


title: CT22 18 hr light

source_name: CT22  18hr light

source_name: Stratagene Reference Rat


title: CT10 6 hr light

source_name: CT10  6hr light

source_name: Stratagene Rat Reference


title: CT16 24 hr night

source_name: CT16  24hr night


title: Night_6hr_pool_v_light_6hr_rat_115

source_name: Individual rat 115

source_name: Pool


title: Night_6hr_pool_v_light_6hr_rat_114

source_name: Individual rat 114

source_name: night 6 hr pool


title: CT10 18 hr night

source_name: CT10  18hr night

source_name: Strategene Rat Reference


title: CT4 48 hr light

source_name: ct4 48 hr


title: CT16 48 hr night

source_name: CT16  48hr night


title: Night_6hr_pool_v_light_6hr_rat_113

source_name: Individual rat

source_name: night Pool 6 hr


title: Night_6hr_pool_v_light_6hr_rat_102

source_name: Individual Rat 102


title: Night_6hr_pool_v_light_6hr_rat_101

source_name: Individual

source_name: pool


title: Night_6hr_pool_v_light_6hr_rat_103


title: Light_6hr_pool_v_night_6hr_rat_139


title: Light_6hr_pool_v_night_6hr_rat_138


title: Light 6 hr pool vs night 6 hr rat 126

source_name: Individual rat 126

source_name: light 6 hr Pool


title: Light_6hr_pool_v_night_6hr_rat_137


title: Light_6hr_pool_v_night_6hr_rat_127

source_name: Individual rat 127


title: 30Mpa, Temp25, 16h-2(low power scan)

status: Public on Apr 14 2005

city: Tsukuba

first: Hitoshi

last: Iwahashi

department: Human Stress Signal Research Center

country: Japan

institute: National Institute of Advanced Industrial Science and Techology

description:
Strains and growth conditions
Saccharomyces cerevisiae strain S288C (mat alpha SUC2 mal mel gal2 CUP1) was grown in YPD medium (2% polypeptone, 1% yeast extract, 2% glucose) at 25°C in pre-cultures for 2-3 days.  This strain was used because the probes on the DNA microarray were produced from S288C as the template for PCR.

Pressure treatment
To achieve an environment with a specific pressure, a 30-11HF4 high pressure vessel (High Pressure Equipment Co. Ltd., USA) with a volume of 500 ml and with a limit of 100 MPa was used.
For pressure treatment, the pre-culture was inoculated into a 50 ml syringe containing YPD medium with a dilution rate of 1000 for the control and 100 for the pressure treatment.  The differential dilution rates were used to obtain cells in the same growth phase after treatment for 16 h.  These syringes were set in the high pressure equipment, pressurized and incubated at 25°C. The control was treated in the same way except for the pressure and dilution rate.  After incubation for 16 h, cells were used for DNA microarray analysis or flow cytometry analysis.

DNA microarray analysis
DNA microarray analysis was carried out with four independent cultures.  Total RNA was isolated by the hot-phenol method.  Poly(A)+RNA was purified from total RNA with Oligotex-dT30 mRNA purification kits (Takara, Kyoto, Japan).  Two to four micrograms of poly (A)+RNA was used for each labeling experiment, and the same amount of each poly (A)+RNA was used on each slide.  The two labeled cDNA pools were mixed and hybridized with a yeast DNA chip (DNA Chip Research, Inc., Yokohama, Japan) for 24-36 h at 65oC.  On this microarray, ORFs of 200-8,000 bp DNA (0.1-0.5 ng) were spotted, and 5880 genes could be analyzed under these conditions.  The details of the microarray procedure and validation studies with our conditions have been described previously.  Detected signals for each ORF were normalized by the intensity dependent (LOWESS) methods (http://www.silicongenetics.com/cgi/SiG.cgi/index.smf). Genes called as induced or repressed were those passing a one sample t-test (p-value cutoff 0.05) and additionally showing more than 2-fold higher or lower expression, respectively, compared to the control. The selected genes were characterized according to the categories of MIPS (Munich Information Center for Protein Sequences, http://mips.gsf.de/).


title: 30Mpa, Temp25, 16h-3(high power scan)


title: 30Mpa, Temp25, 16h-4(high power scan)


title: 30Mpa, Temp25, 16h-3(low power scan)


title: Peptide counts from proteomic analysis of hepatocellular carcinoma WRL 68 cells

status: Public on Apr 07 2005

type: protein

city: Shanghai

first: Zhengjun

last: Chen

state: Shanghai

address: 320 Yueyang Road

country: China

institute: Institute of Biochemistry and Cell Biology

description:
Proteomic analysis of pervanadate-induced tyrosine-phosphorylated proteins in hepatocellular carcinoma WRL 68 cells
Protein tyrosine phosphorylation plays critical roles in modulating biological processes such as cellular proliferation, differentiation, migration, apoptosis and metabolism. To profile tyrosine phosphorylated (pTyr) proteins as well as search novel pTyr proteins as cross-talk points among different cellular pathways, we developed a rapid and efficient approach to identify cellular pTyr proteins and their complexes by a combination of subcellular proteomics approach with signal transduction strategies. Human hepatocytic cells from WRL68 cell line were treated with pervanadate (POV), subfractionated into four fractions and then subjected to immunoaffinity purification with anti-pTyr antibody. The eluted mixtures of the anti-pTyr purification were identified by LC-MS/MS.
Subcellular fractionation and affinity purification of tyrosine-phosphorylated proteins:
WRL68 cells were first grown to 80% confluence in MEM complete medium and then the medium replaced with serum free media. After 15 h, the cells were either untreated or stimulated with 0.1 mM pervanadate (1 mM sodium orthovanadate, 3 mM H2O2) for 10 min. 150-mm cultures of WRL 68 cells were rinsed twice with 4? PBS and then scraped from the dish in 750?l of hypotonic buffer (10 mM Tris, 1 mM NaF, 10 mM IAA, pH 7.5) containing a cocktail of protein inhibitors. After a 20-min incubation on ice, the cells were passed about five times through a 25-g needle. The resulting lysate was subjected to a 15min centrifugation at 1000 rpm at 4?, after which the pellet was resuspended in 250?l of hypotonic buffer and re-extracted by a second round of the trituration and centrifugation. The supernatants of the first and second spins were combined, adjusted to 0.25 M NaCl, and separated into cytosolic (supernatant) and membrane (pellet) fractions bycentrifugation at 19,000 rpm (43,000 g) for 90 min at 4?. All the pellets and total cell lysate were resuspended in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid sodium) containing 1mM pervanadate with a cocktail of protein inhibitors with sonication aid. Cleared cell lysates were incubated overnight at 4? with 30?l monoclonal anti-phosphotyrosine-agrose (Sigma). Precipitated immune complexes were washed three times with 1×HNTG (20 mM HEPES, 150 mM NaCl, 0.1% Triton X-100, 10% Glycerol, pH 7.5) and then eluted with 100 mM phenyl phosphate (Sigma) in lysis buffer at 4?.
Enzyme digestion, mass spectrometry and protein identification:
The sample was digested according to the published method. Chromatography was performed using a surveyor LC system (Thermo Finnigan,SanJose,CA) on C18 reverse phase column(RP, 180 µm x 150 mm, BioBasic® C18, 5 µm, Thermo Hypersil-Keystone). The pump flow rate is split 1:100 for a colum flow rate of 1.5 µL/min.The column effluent is directly electrosprayed using the orthogonal metal needle source without further splitting.Mobile phase A is 0.1% formic acid in water,and the B mobile phase is 0.1% formic acid in acetonitrile. The separation of peptides obtained by enzymatic digest of bile sample was achieved with a gradient of 2-80% B over 360 min.The column effluent from the reverse phase column was analyzed by LCQ Deca XP ion-trap mass spectrometer.The micro-electrospray interface uses a 30 µm metal needle that is orthogonal to the inlet of the LCQ.The mass spectrometer was set that one full MS scan was followed by three MS/MS scans on the three most intense ion from the MS spectrum with the following Dynamic Exclusion™ settings: repeat count, 1; repeat duration, 0.5 min; exclusion duration, 3.0 min. The acquired MS/MS spectra were automatically searched against protein database for human proteins (SWISS-PROT/TrEMBL) using the TurboSEQUEST program in the BioWorks™ 3.0 software suite. An accepted SEQUEST result had to have a ?Cn score of at least 0.1 (regardless of charge state) and Xcorr (one charge?1.5, two charges?2.0, three charges?2.5). Single peptides that alone identify a protein were manually validated after meeting the above criteria.
Bioinformatics analysis:
The pI and MW of the proteins were analyzed using ExPASy proteomics tools accessed from http://cn.expasy.org/tools/#proteome. The grand average hydropathicity (GRAVY) values were determined according to Kyte-Doolittle. The protein subcellular location annotation was from SwissProt &TrEMBL protein database (http://us.expasy.org/sprot/). The protein function family was categorized according to Gene Ontology (GO) annotation terms extracted by InterPro (http://www.ebi.ac.uk./interpro/). The annotation of protein phosphorylation was from SwissProt &TrEMBL protein database (http://us.expasy.org/sprot/) and PhosphoSite (http://www.phosphosite.org). The kinases were annotated according to human kinome.

molecule: protein

source_name: pervanadate-treated hepatocellular carcinoma WRL 68 cells


title: 30Mpa, Temp25, 16h-1(low power scan)


title: 30Mpa, Temp25, 16h-2(high power scan)


title: germ cell tumors (GCT) and normal controls 512A 1

status: Public on Nov 10 2005

city: New York

first: James

last: Korkola

state: NY

address: 1275 York Ave

department: Cell Biology

institute: Memorial Sloan-Kettering

description: Adult Male Germ Cell Tumor

hyb_protocol: standard Affymetrix procedures

scan_protocol: standard Affymetrix procedures

data_processing: RMA (robust multi-array)

label: biotin

source_name: germ cell tumor

label_protocol: Approximately 12 ug of total RNA was processed to produce biotinylated cRNA targets.

extract_protocol: Frozen tissue from a germ cell tumor was minced and homogenized in RLT buffer (Qiagen).Total RNA was extracted from the tissue lysate using an RNeasy kit (Qiagen).

tag: mixed GCT (Yolk Sac, Teratoma)


title: Anaerobic GSNO Chemostat Run 2 Dye swap

status: Public on Apr 06 2005

city: Sheffield

first: Jason

last: Barrett

state: South Yorkshire

address: Firth Court, Western Bank

department: Department of Molecular Biology and Biotechnology

country: United Kingdom

institute: University of Sheffield

description: Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow III Biofermentor under continuous culture (chemostat) conditions. Cells were grown in defined media containing 54 mM glycerol as the sole and limiting source of energy and carbon. The working volume was 1 litre, and the dilution rate 0.2 h-1. In order to establish anaerobic growth, nitrogen was sparged through the chemostat medium prior to inoculation and throughout the course of the experiment at a rate of 0.2 l/min. No dissolved oxygen was detectable using the OxyProbe. Sodium fumarate was added to anaerobic medium at a final concentration of 50 mM to act as a terminal electron acceptor.  Cells were grown as above to steady-state, At steady-state, GSNO was added to the chemostat culture and to the nutrient feed at a final concentration of 200 uM unless otherwise stated. Samples were taken immediately prior to the addition of GSNO and after a period of 5 min exposure to GSNO for subsequent analysis using microarrays. Cells were harvested directly into RNA Protect (Qiagen) to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by the suppliers.

organism: Escherichia coli

source_name: Steady-state continuous cultured experiment samples in presence of 200uM GSNO for 5 minutes

source_name: Steady-state continuous cultured control samples in absence of GSNO


title: Anaerobic GSNO Chemostat Run 2


title: CKX1 BA 15 #4

status: Public on Apr 03 2005

city: Berlin

first: Wolfram

last: Brenner

address: Albrecht-Thaer-Weg 6

department: Applied Genetics

institute: Institute of Biology

description:
Transgenic Arabdopsis thaliana 35S::CKX1 seedlings overexpressing cytokinin oxidase grown in liquid MS were treated for 15 minutes with 6-Benzyl adenine dissolved in diluted KOH.
Total RNA was extracted with the TRIzol method using the high salt precipitation described in the troubleshooting section of the TRIzol manual. The RNA was further purified using Qiagen mini columns. 5 µg of the purified total RNA was used for cDNA synthesis using Invitrogen SuperScriptII reverse transcriptase and a mixture of Ligase, DNA polymerase, and RNase and purified using Qiagen PCR purification columns. The whole amount of cDNA was used as a template to synthesize Biotin-labeled cRNA using the Enzo High Yield RNA transcript labeling kit. After fragmentation as recommended by Affymetrix, the sample was submitted to the RZPD (Berlin), where the subsequent steps were performed to generate a table containing the normalized expression values and expression calls.

source_name: Seedlings treated with 6-Benzyl adenine dissolved in diluted KOH for 15 min


title: Anaerobic GSNO Chemostat Run 1 Dye Swap


title: Anaerobic GSNO Chemostat Run 1


title: 8 (Cy3) - 16 (Cy5)

source_name: RAT # 16

source_name: RAT # 8


title: CKX1 BA 15 #3


title: Aerobic GSNO Chemostat 2 Run 2

description: Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow III Biofermentor under continuous culture (chemostat) conditions. Cells were grown in defined media containing 54 mM glycerol as the sole and limiting source of energy and carbon. The working volume was 1 litre, and the dilution rate 0.2 h-1. For aerobic growth, the air-flow rate was 1 l/min, and the dissolved oxygen tension was maintained at 40% air saturation by measuring oxygen dissolved in the culture using a Broadley James D140 OxyProbe® electrode and automated adjustment of stirring rate. Cells were grown as above to steady-state, At steady-state, GSNO was added to the chemostat culture and to the nutrient feed at a final concentration of 200 uM unless otherwise stated. Samples were taken immediately prior to the addition of GSNO and after a period of 5 min exposure to GSNO for subsequent analysis using microarrays. Cells were harvested directly into RNA Protect (Qiagen) to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by the suppliers.


title: Aerobic GSNO Chemostat Run 2


title: tumor.5

status: Public on Mar 22 2005

city: Chicago

first: Simon

last: Lin

state: IL

department: Bioinformatics Core

institute: Northwestern University

description:
ANIMALS

Patched heterozygous mice (Goodrich et al., 1997) were obtained from Matthew Scott’s lab at Stanford, and maintained by breeding with 129X1/SvJ mice from Jackson Laboratories. Math1-GFP transgenic mice (Lumpkin et al., 2003) were provided by Jane Johnson at UT Southwestern Medical Center. Math1-GFP/patched+/- mice were generated by crossing patched heterozygotes with Math1-GFP mice, and then backcrossing to Math1-GFP mice three times before further analysis. All mice were maintained in the Cancer Center Isolation Facility at Duke University Medical Center.

ISOLATION OF CELLS

Granule cell precursors (GCPs) were isolated from 7-day-old (P7) patched+/- mice; preneoplastic cells were obtained from 6 week-old patched mutants; and tumor cells were obtained from 10-25-week old patched mutants displaying physical and behavioral signs of medulloblastoma. Cells were isolated from each source using a protocol described in (Wechsler- Reya and Scott, 1999). Briefly, cerebella were digested in solution containing 10 U/ml papain Oliver et al. Pre-Neoplastic Stage of Medulloblastoma - 8 - (Worthington, Lakewood, NJ) and 250 U/ml DNase (Sigma) and triturated to obtain a cell suspension. This suspension was centrifuged through a step gradient of 35% and 65% Percoll (Amersham Biosciences, Piscataway, NJ), and cells were harvested from the 35%-65% interface. Cells were resuspended in serum-free culture medium consisting of Neurobasal containing B27 supplement, sodium pyruvate, L-glutamine, and penicillin/streptomycin (all from Invitrogen, Carlsbad, California) and counted on a hemacytometer. Cells used for RNA isolation were centrifuged and flash frozen in liquid nitrogen. For proliferation assays or immunostaining, cells were plated on poly-D-lysine (PDL)-coated tissue culture vessels and incubated in serum-free culture medium.

MICROARRAY HYBRIDIZATION AND ANALYSIS

RNA from GCPs, pre-neoplastic cells and tumor cells (isolated as described above, but not FACS-sorted) and from normal adult cerebellum (not dissociated) was converted to cDNA using the Superscript Choice cDNA kit (Invitrogen) and a T7-dT(24) primer (Genset/Proligo, Boulder, CO). cRNA was generated using a T7-transcription/labeling kit from Enzo Life Sciences and hybridized to Affymetrix U74Av2 chips (Affymetrix, Santa Clara, CA). Chips were scanned, and hybridization data were acquired using Affymetrix Suite 5.0 software. Affymetrix CEL files were normalized and quantified using Bioconductor software with the gcRMA model to quantify gene expression levels (Gentleman and Carey, 2002). Unsupervised principal components analysis (PCA) was used to identify the relationships among normal adult cerebellum, GCPs, pre-neoplastic cells and tumor cells based on expression profiles. To identify genes that were differentially expressed among GCPs, pre-neoplastic cells and tumor cells, supervised analysis was carried out. A gene-by-gene ANOVA model with three groups (GCP, pre-neoplastic, tumor) was used to fit the log2-transformed intensities. To correct for multiple comparisons, the nominal p-value was adjusted using the false discovery rate (Benjamini and Hochberg, 1995). Genes were considered to be differentially expressed if they satisfied all of the following criteria: a difference in expression greater than 1.9-fold between any two groups, a maximum absolute intensity difference larger than 32 units, and an adjusted pvalue < 0.01. There were 118 genes that met these criteria. The identities of differentially expressed genes were verified by integrating data from the Affymetrix and Unigene databases. Gene functions were determined using information from Gene Ontology, Unigene, LocusLink and PubMed databases. Clustering was performed with Cluster and Treeview (Eisen et al., 1998). All statistical analysis was performed using R-1.7 software (Dalgaard, 2002). Results were visualized with Spotfire 6.0 (Somerville, MA).

CORRESPONDING AUTHOR

Robert J. Wechsler-Reya*
Dept. of Pharmacology & Cancer Biology,
Duke University Medical Center Box 3813, Durham, NC 27710
Phone: 919-613-8754. Fax: 919-668-3556. Email: rw.reya@duke.edu
Keywords = medulloblastoma
Keywords = brain tumor

source_name: tumor


title: tumor.4


title: tumor.2


title: 30Mpa, Temp25, 16h-4(low power scan)


title: CKX1 BA 15 #2


title: Col-0 BA 120 #2

description:
Arabdopsis thaliana Col-0 seedlings grown for 6 days in liquid MS were treated for 120 minutes with 6-Benzyl adenine dissolved in diluted KOH.
Total RNA was extracted with the TRIzol method using the high salt precipitation described in the troubleshooting section of the TRIzol manual. The RNA was further purified using Qiagen mini columns. 5 µg of the purified total RNA was used for cDNA synthesis using Invitrogen SuperScriptII reverse transcriptase and a mixture of Ligase, DNA polymerase, and RNase and purified using Qiagen PCR purification columns. The whole amount of cDNA was used as a template to synthesize Biotin-labeled cRNA using the Enzo High Yield RNA transcript labeling kit. After fragmentation as recommended by Affymetrix, the sample was submitted to the RZPD (Berlin), where the subsequent steps were performed resulting in a table containing the normalized expression values and expression calls.

source_name: Seedlings treated with 6-Benzyl adenine dissolved in diluted KOH for 120 min


title: Col-0 BA 15 #2

description:
Arabdopsis thaliana Col-0 seedlings grown for 6 days in liquid MS were treated for 15 minutes with 6-Benzyl adenine dissolved in diluted KOH.
Total RNA was extracted with the TRIzol method using the high salt precipitation described in the troubleshooting section of the TRIzol manual. The RNA was further purified using Qiagen mini columns. 5 µg of the purified total RNA was used for cDNA synthesis using Invitrogen SuperScriptII reverse transcriptase and a mixture of Ligase, DNA polymerase, and RNase and purified using Qiagen PCR purification columns. The whole amount of cDNA was used as a template to synthesize Biotin-labeled cRNA using the Enzo High Yield RNA transcript labeling kit. After fragmentation as recommended by Affymetrix, the sample was submitted to the RZPD (Berlin), where the subsequent steps were performed to generate a table containing the normalized expression values and expression calls.


title: CKX1 BA 0 #3

description:
Transgenic Arabdopsis thaliana 35S::CKX1 seedlings overexpressing cytokinin oxidase grown in liquid MS were treated for 2 hours with the solvent used for dissolving Benzyl adenin in the induction experiments.
Total RNA was extracted with the TRIzol method using the high salt precipitation described in the troubleshooting section of the TRIzol manual. The RNA was further purified using Qiagen mini columns. 5 µg of the purified total RNA was used for cDNA synthesis using Invitrogen SuperScriptII reverse transcriptase and a mixture of Ligase, DNA polymerase, and RNase and purified using Qiagen PCR purification columns. The whole amount of cDNA was used as a template to synthesize Biotin-labeled cRNA using the Enzo High Yield RNA transcript labeling kit. After fragmentation as recommended by Affymetrix, the sample was submitted to the RZPD (Berlin), where the subsequent steps were performed to generate a table containing the normalized expression values and expression calls.

source_name: Seedlings treated with control solution


title: Tiling microarray analysis of rice indica chromosome 10(reverse strand, normal)

status: Public on Apr 09 2005

city: New Haven

first: Xing-Wang

last: Deng

address: 165 Prospect

department: MCD Biology

institute: Yale University

description: The four RNA population from seedling roots, seedling shoots, panicles, and suspension-cultured cells were pooled in equal amount and cDNAs were derived from the RNA mixture. Total RNA and mRNA were isolated using the RNeasy Plant Mini kit (Qiagen, Valencia, CA) and the Oligotex mRNA kit (Qiagen), respectively. First-strand cDNA was generated from 4 µg poly(A)+ RNA with SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). The cDNAs were precipitated in ethanol:isopropanol (1:1 v/v) and resuspended in 0.1 M NaHCO3 to facilitate coupling of Alexa Fluor 555 NHS esters (Molecular Probes, Eugene, OR) to the reactive groups of the amino-allyl dUTPs and were purified with CyScribe GFX glass fiber spin columns (Amersham Bioscience, Piscataway, NJ). Microarrays were hybridized with 4 µg labeled cDNA in 300 µL hybridization buffer (50 mM MES, 0.5 M NaCl, 10 mM EDTA, and 0.005% (v/v) Tween-20) for 16 hours at 50°C in disposable adhesive chambers (Grace BioLabs, Bend, OR) in a hybridization oven with constant agitation. Hybridized arrays were washed on an orbital platform in non-stringent buffer (6× SSPE, 0.01% [v/v] Tween-20) for 10 minutes at room temperature, then in stringent buffer (100 mM MES, 0.1 M NaCl, 0.01% Tween-20) for 30 minutes at 45°C. This was followed by a 5-minute wash in non-stringent buffer and a 2-minute wash in 0.1X SSC. The arrays were dried with compressed nitrogen and scanned with an Axon 4000B laser scanner at 5 µm resolution. The fluorescence intensity data were extracted with the NimbleScan software (NimbleGen Systems, Madison, WI).

organism: Oryza sativa

source_name: Poly(A)+ RNA derived from rice seedling roots, seedling shoots, panicles, and suspension cultured cells.


title: CKX1 BA 0 #2


title: Aerobic GSNO Chemostat 2 Run 1


title: CKX1 BA 15 #1


title: Control 7-12-02

source_name: Yeast BY4730


title: Tiling microarray analysis of rice indica chromosome 10(forward strand, normal)


title: Tiling microarray analysis of rice japonica chromosome 10 reverse strand normal

description:
The four RNA population from seedling roots, seedling shoots, panicles, and suspension-cultured cells were pooled in equal amount and cDNAs were derived from the RNA mixture. Total RNA and mRNA were isolated using the RNeasy Plant Mini kit (Qiagen, Valencia, CA) and the Oligotex mRNA kit (Qiagen), respectively. First-strand cDNA was generated from 4 µg poly(A)+ RNA with SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). The cDNAs were precipitated in ethanol:isopropanol (1:1 v/v) and resuspended in 0.1 M NaHCO3 to facilitate coupling of Alexa Fluor 555 NHS esters (Molecular Probes, Eugene, OR) to the reactive groups of the amino-allyl dUTPs and were purified with CyScribe GFX glass fiber spin columns (Amersham Bioscience, Piscataway, NJ). Microarrays were hybridized with 4 µg labeled cDNA in 300 µL hybridization buffer (50 mM MES, 0.5 M NaCl, 10 mM EDTA, and 0.005% (v/v) Tween-20) for 16 hours at 50°C in disposable adhesive chambers (Grace BioLabs, Bend, OR) in a hybridization oven with constant agitation. Hybridized arrays were washed on an orbital platform in non-stringent buffer (6× SSPE, 0.01% [v/v] Tween-20) for 10 minutes at room temperature, then in stringent buffer (100 mM MES, 0.1 M NaCl, 0.01% Tween-20) for 30 minutes at 45°C. This was followed by a 5-minute wash in non-stringent buffer and a 2-minute wash in 0.1X SSC. The arrays were dried with compressed nitrogen and scanned with an Axon 4000B laser scanner at 5 µm resolution. The fluorescence intensity data were extracted with the NimbleScan software (NimbleGen Systems, Madison, WI).
Keywords = Rice
Keywords = poly(A)+ RNA


title: Col-0 BA 15 #1


title: CKX1 BA 0 #1


title: Aerobic GSNO Chemostat Run 1


title: tumor.3