title: GCP.3

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).

Keywords = cerebellar granule cell precursors (GCPs)

source_name: GCP


title: succinate2

status: Public on Mar 11 2005

type: RNA

city: Madison

first: Mingzhu

last: Liu

state: WI

country: USA

institute: University of Wisconsin-Madison

description:
The study investigated the effect of different carbon sources on E. coli global gene expression. We grew MG1655 cells aerobically in MOPS minimal medium with either glucose, glycerol, succinate, L-alanine, acetate, or L-proline as the carbon supply. Samples were taken from each culture at mid log phase and were RNA-stabilized using Qiagen RNAProtect Bacterial Reagent (Qiagen). Total RNA was then isolated using MasterPure kits (Epicentre Technologies). Purified RNA was reverse-transcribed to cDNA, labeled and hybridized to Affymetrix GeneChipÒ E. coli Antisense Genome Arrays as recommended in the technical manual (www.affymetrix.com).
This sample is from succinate-grown cells.

molecule: total RNA

organism: Escherichia coli

source_name: E. coli cell


title: 30Mpa, Temp25, 16h-1(high 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/).

organism: Saccharomyces cerevisiae


title: succinate1


title: Tiling microarray analysis of rice japonica chromosome 10(forward strand)

status: Public on Apr 09 2005

city: New Haven

first: Xing-Wang

last: Deng

state: CT

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).
Keywords = Rice
Keywords = poly(A)+ RNA

organism: Oryza sativa

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


title: pretumor.4

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).

organism: Mus musculus

source_name: pretumor


title: pretumor.2

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).
Keywords = medulloblastoma
Keywords = pre-neoplastic


title: glucose2

description:
The study investigated the effect of different carbon sources on E. coli global gene expression. We grew MG1655 cells aerobically in MOPS minimal medium with either glucose, glycerol, succinate, L-alanine, acetate, or L-proline as the carbon supply. Samples were taken from each culture at mid log phase and were RNA-stabilized using Qiagen RNAProtect Bacterial Reagent (Qiagen). Total RNA was then isolated using MasterPure kits (Epicentre Technologies). Purified RNA was reverse-transcribed to cDNA, labeled and hybridized to Affymetrix GeneChipÒ E. coli Antisense Genome Arrays as recommended in the technical manual (www.affymetrix.com).
This sample is from glucose-grown cells.


title: pretumor.1


title: glycerol1

description:
The study investigated the effect of different carbon sources on E. coli global gene expression. We grew MG1655 cells aerobically in MOPS minimal medium with either glucose, glycerol, succinate, L-alanine, acetate, or L-proline as the carbon supply. Samples were taken from each culture at mid log phase and were RNA-stabilized using Qiagen RNAProtect Bacterial Reagent (Qiagen). Total RNA was then isolated using MasterPure kits (Epicentre Technologies). Purified RNA was reverse-transcribed to cDNA, labeled and hybridized to Affymetrix GeneChipÒ E. coli Antisense Genome Arrays as recommended in the technical manual (www.affymetrix.com).
This sample is from glycerol-grown cells.


title: glucose3


title: GCP.4


title: tumor.1

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: pretumor.5


title: glucose5


title: acetate2

description:
The study investigated the effect of different carbon sources on E. coli global gene expression. We grew MG1655 cells aerobically in MOPS minimal medium with either glucose, glycerol, succinate, L-alanine, acetate, or L-proline as the carbon supply. Samples were taken from each culture at mid log phase and were RNA-stabilized using Qiagen RNAProtect Bacterial Reagent (Qiagen). Total RNA was then isolated using MasterPure kits (Epicentre Technologies). Purified RNA was reverse-transcribed to cDNA, labeled and hybridized to Affymetrix GeneChipÒ E. coli Antisense Genome Arrays as recommended in the technical manual (www.affymetrix.com).
This sample is from acetate-grown cells.


title: alanine2

description:
The study investigated the effect of different carbon sources on E. coli global gene expression. We grew MG1655 cells aerobically in MOPS minimal medium with either glucose, glycerol, succinate, L-alanine, acetate, or L-proline as the carbon supply. Samples were taken from each culture at mid log phase and were RNA-stabilized using Qiagen RNAProtect Bacterial Reagent (Qiagen). Total RNA was then isolated using MasterPure kits (Epicentre Technologies). Purified RNA was reverse-transcribed to cDNA, labeled and hybridized to Affymetrix GeneChipÒ E. coli Antisense Genome Arrays as recommended in the technical manual (www.affymetrix.com).
This sample is from alanine-grown cells.


title: glucose1


title: GCP.1


title: proline1

description:
The study investigated the effect of different carbon sources on E. coli global gene expression. We grew MG1655 cells aerobically in MOPS minimal medium with either glucose, glycerol, succinate, L-alanine, acetate, or L-proline as the carbon supply. Samples were taken from each culture at mid log phase and were RNA-stabilized using Qiagen RNAProtect Bacterial Reagent (Qiagen). Total RNA was then isolated using MasterPure kits (Epicentre Technologies). Purified RNA was reverse-transcribed to cDNA, labeled and hybridized to Affymetrix GeneChipÒ E. coli Antisense Genome Arrays as recommended in the technical manual (www.affymetrix.com).
This sample is from proline-grown cells.


title: alanine1


title: RAT_Control2

status: Public on Mar 08 2005

city: Baltimore

first: Shwu-Fan

last: Ma

state: MD

address: 5501 Hopkins Bayview Circle

institute: Johns Hopkins University

description:
Rats (n = 2) were mechanically ventilated (12 ml/kg, 5 hrs) with room air and control group (n = 2) spontaneously respiring through identical tracheal cannulas without mechanical ventilation.
Sample_age: 8 weeks old
Sample_hybridization: total RNA was converted to first-strand cDNA using a hybrid reverse transcription primer consisting of oligo-dT and T7 RNA polymerase promoter sequences. The single-stranded cDNA was then converted to double-stranded cDNA. Complementary DNA corresponding to 5-10 µg of total RNA was used in a cRNA amplification step using T7 RNA polymerase and two biotinylated nucleotide precursors. The resulting biotinylated cRNA was fragmented to a size of approximately 50 bp, and approximately 20-30 µg of the biotinylated cRNA was hybridized to the RG_U34A GeneChip (Affymetrix, Santa Clara, CA). The bound cRNA was visualized by binding of streptavidin/phycoerythrin conjugates to the hybridized GeneChip, followed by laser scanning of bound phycoerythrin.
Sample_RNA_Isolation: Tissues (~50 mg) were taken frozen and directly solubilized in chaotropic solubilization buffer using a Brinkman Polytron tissue disruptor. Larger tissue fragments (>100mg) were pulverized into frozen powder with a mortar and pestle, pre-chilled to liquid nitrogen temperature, and then the frozen powder was solubilized with the Polytron. RNA was purified using Trizol LS (Life Technologies) and an additional RNA purification step was conducted using the RNAeasy purification kit (Qiagen Inc., Valencia, CA). Approximately 10 µg of purified, total RNA was used for analyses.
Sample_sex: male
Sample_strain: Wistar
Keywords = Rat, Bioinformatics, mechanical ventilation, early stress response genes, LPS

organism: Rattus norvegicus

source_name: rat lungs


title: GCP.2


title: Control2

description:
Mice were exposed to large tidal volume (17 ml/kg, 6 hrs or 35 ml/kg, 2 hrs) using a small animal mechanical ventilator with non-mechanical ventilated, spontaneous breathing mice used as a control (n=2).
Sample_age: 7-8 weeks old
Sample_hybridization: total RNA was converted to first-strand cDNA using a hybrid reverse transcription primer consisting of oligo-dT and T7 RNA polymerase promoter sequences. The single-stranded cDNA was then converted to double-stranded cDNA. Complementary DNA corresponding to 5-10 µg of total RNA was used in a cRNA amplification step using T7 RNA polymerase and two biotinylated nucleotide precursors. The resulting biotinylated cRNA was fragmented to a size of approximately 50 bp, and approximately 20-30 µg of the biotinylated cRNA was hybridized to the MG_U74Av2 GeneChip (Affymetrix, Santa Clara, CA). The bound cRNA was visualized by binding of streptavidin/phycoerythrin conjugates to the hybridized GeneChip, followed by laser scanning of bound phycoerythrin.
Sample_RNA_Isolation: Tissues (~50 mg) were taken frozen and directly solubilized in chaotropic solubilization buffer using a Brinkman Polytron tissue disruptor. Larger tissue fragments (>100mg) were pulverized into frozen powder with a mortar and pestle, pre-chilled to liquid nitrogen temperature, and then the frozen powder was solubilized with the Polytron. RNA was purified using Trizol LS (Life Technologies) and an additional RNA purification step was conducted using the RNAeasy purification kit (Qiagen Inc., Valencia, CA). Approximately 10 µg of purified, total RNA was used for analyses.
Sample_sex: male
Sample_strain: C57BL6
Keywords = Murine
Keywords = Bioinformatics
Keywords = mechanical ventilation
Keywords = early stress response gene
Keywords = LPS

source_name: murine lungs


title: Col-0 BA 120 #1

status: Public on Apr 03 2005

city: Berlin

first: Wolfram

last: Brenner

address: Albrecht-Thaer-Weg 6

department: Applied Genetics

country: Germany

institute: Institute of Biology

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.

organism: Arabidopsis thaliana

source_name: Seedlings treated with control solution


title: glucose4


title: Control1


title: Col-0 BA 0 #1

description:
Arabdopsis thaliana Col-0 seedlings 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.


title: glycerol2


title: RAT_MV2


title: MV2


title: RAT_Control1


title: acetate1


title: Lung slide3

status: Public on Mar 05 2005

city: Ames

first: Christopher

middle: K

last: Tuggle

state: IA

address: 2255 Kildee Hall

department: Animal Science

institute: Iowa State University

description:
Total RNAs from control (3 pigs), 24 hour (3 pigs) and 48 hour (3 pigs) porcine lungs after intranasal Salmonella Choleraesuis infection were isolated by RNeasy kit and treated by DNaseI. For generating amino-allyl-dUTP labeled single strand cDNA, 20 ug RNA from the control and infected lungs were reverse transcribed for 2-3 h at 42C in the presence of 6 ug random and 1 ug oligo-dT(18) primers (Invitrogen/Life Technologies), 1 X first strand buffer, 10mM DTT, dNTP (25 mM dCTP, 25 mM dGTP, 25 mM dATP, 15 mM dTTP, 10 mM aa-dUTP), and 400 units of Superscript II reverse transcriptase (Invitrogen/Life Technologies). RNA was hydrolyzed with 1M NaOH, and then neutralized with 1M HEPES (pH 7.0). First-strand cDNA was precipitated, air dried and re-suspended with 4 ul 0.1M Na2CO3 buffer (pH 9.0), mixed with either Cy3 or Cy5 NHS-ester (Amersham Pharmacia), and incubated 1 hr at room temperature. Cy3 and Cy5 labeled cDNA were purified on QIAquick PCR purification columns (Qiagen, Valencia, CA) according to manufacturer’s instructions, except that QIAquick wash buffer was replaced by phosphate buffer. Eluted cDNA were precipitated by ETOH and hybridized to the Qiagen-NRSP-8 porcine oligonucleotide microarray at 42C for 12-16 hr in hybridization solution containing 50% formamide, 5 X SSC, 0.1% SDS and 0.2 ug/ul sheared salmon sperm DNA. After hybridization, microarray slides were washed by 2 X SSC, 0.1% SDS for 5 min at 42C, 0.1 X SSC for 1 min at room temperature for four times, and 0.01 X SSC for 10 sec at room temperature, and dried by centrifugation.Slides were scanned at 10 um resolution using a ScanArray 5000 scanner under conditions to limit saturation to <1% and were saved as TIFF images. The intensities of spots on each image were quantified by ImaGene 5.1 software, and data were saved as .txt files for further analyses.
Keywords = pig
Keywords = microarray
Keywords = lung
Keywords = gene expression
Keywords = infection

organism: Sus scrofa

source_name: Lung sample from 48 hour intranasal infected pig (pig ID 8)

source_name: Lung sample from day -3 control pig (pig ID 19)


title: Lung slide2

description:
Total RNAs from control (3 pigs), 24 hour (3 pigs) and 48 hour (3 pigs) porcine lungs after intranasal Salmonella Choleraesuis infection were isolated by RNeasy kit and treated by DNaseI. For generating amino-allyl-dUTP labeled single strand cDNA, 20 ug RNA from the control and infected lungs were reverse transcribed for 2-3 h at 42C in the presence of 6 ug random and 1 ug oligo-dT(18) primers (Invitrogen/Life Technologies), 1 X first strand buffer, 10mM DTT, dNTP (25 mM dCTP, 25 mM dGTP, 25 mM dATP, 15 mM dTTP, 10 mM aa-dUTP), and 400 units of Superscript II reverse transcriptase (Invitrogen/Life Technologies). RNA was hydrolyzed with 1M NaOH, and then neutralized with 1M HEPES (pH 7.0). First-strand cDNA was precipitated, air dried and re-suspended with 4 ul 0.1M Na2CO3 buffer (pH 9.0), mixed with either Cy3 or Cy5 NHS-ester (Amersham Pharmacia), and incubated 1 hr at room temperature. Cy3 and Cy5 labeled cDNA were purified on QIAquick PCR purification columns (Qiagen, Valencia, CA) according to manufacturer’s instructions, except that QIAquick wash buffer was replaced by phosphate buffer. Eluted cDNA were precipitated by ETOH and hybridized to the Qiagen-NRSP-8 porcine oligonucleotide microarray at 42C for 12-16 hr in hybridization solution containing 50% formamide, 5 X SSC, 0.1% SDS and 0.2 ug/ul sheared salmon sperm DNA. After hybridization, microarray slides were washed by 2 X SSC, 0.1% SDS for 5 min at 42C, 0.1 X SSC for 1 min at room temperature for four times, and 0.01 X SSC for 10 sec at room temperature, and dried by centrifugation.Slides were scanned at 10 um resolution using a ScanArray 5000 scanner under conditions to limit saturation to <1% and were saved as TIFF images. The intensities of spots on each image were quantified by ImaGene 5.1 software, and data were saved as .txt files for further analyses.

Keywords = pig
Keywords = microarray
Keywords = lung
Keywords = gene expression
Keywords = infection

source_name: Lung sample from 24 hour intranasal infected pig (pig ID 13)


title: Tissue slide23

description:
Total RNA from different porcine tissues, lung, muscle (ham), liver, and small intestine (duodenum; approximately 5 cm from stomach), were isolated by RNeasy kit and treated by DNaseI. For generating amino-allyl-dUTP labeled single strand cDNA, 30 ug RNA from the four tissue samples were reverse transcribed for 2-3 h at 42C in the presence of 6 ug random and 1 ug oligo-dT(18) primers (Invitrogen/Life Technologies), 1 X first strand buffer, 10mM DTT, dNTP (25 mM dCTP, 25 mM dGTP, 25 mM dATP, 15 mM dTTP, 10 mM aa-dUTP), and 400 units of Superscript II reverse transcriptase (Invitrogen/Life Technologies). RNA was hydrolyzed with 1M NaOH, and then neutralized with 1M HEPES (pH 7.0). First-strand cDNA was precipitated, air dried and re-suspended with 4 ul 0.1M Na2CO3 buffer (pH 9.0), mixed with either Cy3 or Cy5 NHS-ester (Amersham Pharmacia), and incubated 1 hr at room temperature. Cy3 and Cy5 labeled cDNA were purified on QIAquick PCR purification columns (Qiagen, Valencia, CA) according to manufacturer’s instructions, except that QIAquick wash buffer was replaced by phosphate buffer. Eluted cDNA were precipitated by ETOH and hybridized to the Qiagen-NRSP-8 porcine oligonucleotide microarray at 42C for 12-16 hr in hybridization solution containing 50% formamide, 5 X SSC, 0.1% SDS and 0.2 ug/ul sheared salmon sperm DNA. After hybridization, microarray slides were washed by 2 X SSC, 0.1% SDS for 5 min at 42C, 0.1 X SSC for 1 min at room temperature for four times, and 0.01 X SSC for 10 sec at room temperature, and dried by centrifugation.Slides were scanned at 10 um resolution using a ScanArray 5000 scanner under conditions to limit saturation to <1% and were saved as TIFF images. The intensities of spots on each image were quantified by ImaGene 5.1 software, and data were saved as .txt files for further analyses.
Keywords = pig
Keywords = microarray
Keywords = tissue
Keywords = gene expression

source_name: Pig 415 liver

source_name: Pig 415 small intestine


title: Lung slide4

source_name: Lung sample from day -3 control pig (pig ID 22)

source_name: Lung sample from 24 hour intranasal infected pig (pig ID 10)


title: Tissue slide22

source_name: Pig 415 lung


title: Lung slide8

description: Total RNAs from control (3 pigs), 24 hour (3 pigs) and 48 hour (3 pigs) porcine lungs after intranasal Salmonella Choleraesuis infection were isolated by RNeasy kit and treated by DNaseI. For generating amino-allyl-dUTP labeled single strand cDNA, 20 ug RNA from the control and infected lungs were reverse transcribed for 2-3 h at 42C in the presence of 6 ug random and 1 ug oligo-dT(18) primers (Invitrogen/Life Technologies), 1 X first strand buffer, 10mM DTT, dNTP (25 mM dCTP, 25 mM dGTP, 25 mM dATP, 15 mM dTTP, 10 mM aa-dUTP), and 400 units of Superscript II reverse transcriptase (Invitrogen/Life Technologies). RNA was hydrolyzed with 1M NaOH, and then neutralized with 1M HEPES (pH 7.0). First-strand cDNA was precipitated, air dried and re-suspended with 4 ul 0.1M Na2CO3 buffer (pH 9.0), mixed with either Cy3 or Cy5 NHS-ester (Amersham Pharmacia), and incubated 1 hr at room temperature. Cy3 and Cy5 labeled cDNA were purified on QIAquick PCR purification columns (Qiagen, Valencia, CA) according to manufacturer’s instructions, except that QIAquick wash buffer was replaced by phosphate buffer. Eluted cDNA were precipitated by ETOH and hybridized to the Qiagen-NRSP-8 porcine oligonucleotide microarray at 42C for 12-16 hr in hybridization solution containing 50% formamide, 5 X SSC, 0.1% SDS and 0.2 ug/ul sheared salmon sperm DNA. After hybridization, microarray slides were washed by 2 X SSC, 0.1% SDS for 5 min at 42C, 0.1 X SSC for 1 min at room temperature for four times, and 0.01 X SSC for 10 sec at room temperature, and dried by centrifugation.Slides were scanned at 10 um resolution using a ScanArray 5000 scanner under conditions to limit saturation to <1% and were saved as TIFF images. The intensities of spots on each image were quantified by ImaGene 5.1 software, and data were saved as .txt files for further analyses.

source_name: Lung sample from day -3 control pig (pig ID 16)

source_name: Lung sample from 48 hour intranasal infected pig (pig ID 5)


title: Lung slide7

source_name: Lung sample from 24 hour intranasal infected pig (pig ID 6)


title: Tissue slide21

description:
Total RNA from different porcine tissues, lung, muscle (ham), liver, and small intestine (duodenum; approximately 5 cm from stomach), were isolated by RNeasy kit and treated by DNaseI. For generating amino-allyl-dUTP labeled single strand cDNA, 30 ug RNA from the four tissue samples were reverse transcribed for 2-3 h at 42C in the presence of 6 ug random and 1 ug oligo-dT(18) primers (Invitrogen/Life Technologies), 1 X first strand buffer, 10mM DTT, dNTP (25 mM dCTP, 25 mM dGTP, 25 mM dATP, 15 mM dTTP, 10 mM aa-dUTP), and 400 units of Superscript II reverse transcriptase (Invitrogen/Life Technologies). RNA was hydrolyzed with 1M NaOH, and then neutralized with 1M HEPES (pH 7.0). First-strand cDNA was precipitated, air dried and re-suspended with 4 ul 0.1M Na2CO3 buffer (pH 9.0), mixed with either Cy3 or Cy5 NHS-ester (Amersham Pharmacia), and incubated 1 hr at room temperature. Cy3 and Cy5 labeled cDNA were purified on QIAquick PCR purification columns (Qiagen, Valencia, CA) according to manufacturer’s instructions, except that QIAquick wash buffer was replaced by phosphate buffer. Eluted cDNA were precipitated by ETOH and hybridized to the Qiagen-NRSP-8 porcine oligonucleotide microarray at 42C for 12-16 hr in hybridization solution containing 50% formamide, 5 X SSC, 0.1% SDS and 0.2 ug/ul sheared salmon sperm DNA. After hybridization, microarray slides were washed by 2 X SSC, 0.1% SDS for 5 min at 42C, 0.1 X SSC for 1 min at room temperature for four times, and 0.01 X SSC for 10 sec at room temperature, and dried by centrifugation.Slides were scanned at 10 um resolution using a ScanArray 5000 scanner under conditions to limit saturation to <1% and were saved as TIFF images. The intensities of spots on each image were quantified by ImaGene 5.1 software, and data were saved as .txt files for further analyses.

Keywords = pig
Keywords = microarray
Keywords = tissue
Keywords = gene expression

source_name: Pig 415 muscle


title: Lung slide6

source_name: Lung sample from 48 hour intranasal infected pig (pig ID 12)


title: Lung slide9


title: Tissue slide18

source_name: Pig 404 liver

source_name: Pig 404 muscle


title: proline2

source_name: E. coli cell


title: Tissue slide24


title: Tissue slide16

source_name: Pig 411 small intestine

source_name: Pig 411 liver


title: Lung slide5


title: Comparing between control and neural induced ectodermal cell, set2_sample2

city: Irvine

first: Yongchol

last: Shin

state: CA

address: 4115 McGaugh Hall

department: Developmental and Cell Biology

institute: University of California, Irvine

description:
Two-cell stage embryos were microinjected with 4 ng of dominant negative BMP receptor I (DNBR) mRNA.  Animal cap explants were isolated from blastula stage (st 8-8.5) embryos injected with DNBR mRNA and control uninjected embryos, and then allowed to develop until early gastrula stage (st 10.5). 50 micrograms of total RNA isolated from the explants was used in reverse transcription reactions with oligo(dT)20 using Superscript II enzyme (Gibco) in the presence of Cy3- and Cy5-dUTP.  RNA from animal caps injected with DNBR mRNA was labeled with Cy5–dUTP, and control RNA with Cy3-dUPT. The samples were treated with 1M NaOH to degrade the RNA.  Cy3 and Cy5 fluorescent labeled probes were combined, purified first using a PCR purification kit (Qiagen), then using Microcon YM-30 spin column filters (Amicon) to remove unincorporated dyes and free nucleotides.  The eluated solution volume was mixed with an equal volume of 2x hybridization buffer [0.2% SDS, 6.8x SSC and yeast tRNA at 1 ug/ul].  Hybridization was performed at 65C for 20 hours. Hybridized slides were scanned with a GenePix 4000B scanner (Axon Instruments). The laser power was set to 100% and PMT voltage (normally 700-900 volts) was adjusted depending upon levels of signal saturation.  For array data analysis, we used a GenePix Pro image acquisition program, a Cyber-T program (http://visitor.ics.uci.edu/genex/cybert/) and GeneData Expressionist (GeneData).
Keywords = neural induction
Keywords = BMP
Keywords = nervous system
Keywords = Xenopus
Keywords = microarray

organism: Xenopus laevis

source_name: Ectodermal cells those were converted to neuroectodermal cells by inhibition of BMP signalig

source_name: Control ectodermal cells


title: Tissue slide15

source_name: Pig 411 muscle

source_name: Pig 411 lung


title: pretumor.3

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).
Keywords = medulloblastoma
Keywords = pre-neoplastic

source_name: pretumor


title: RAT_MV1

source_name: rat lungs


title: MV1

source_name: murine lungs


title: Tissue slide17

source_name: Pig 404 lung


title: Lung slide1

source_name: Day -3 control lung (Pig ID 19)

source_name: lung sample after 24 hour intranasal Salmonella infection  (pig ID 13)


title: Tissue slide19

source_name: Pig 404 small intestine


title: Tissue slide20

source_name: pig 404 lung


title: RA responsive genes 3

description:
Embryos were treated in groups of 25 in 60 mm petri dishes with 10 ml of 0.1x MBS containing 10-6 M of RAR antagonist (AGN 193109), 10-6 M RAR agonist (TTNPB), or vehicle control (0.1% v/v ethanol) from stage 7 to stage 11 at 16-19°C. Total embryo RNA was isolated using Trizol reagent (InVitrogen Life Technologies), DNAse treated and LiCl precipitated. Poly A+ RNA was isolated using OligoTex (Qiagen).
Keywords = retinoid
Keywords = microarray
Keywords = RAR
Keywords = neurula
Keywords = anteroposterior patterning
Keywords = and organogenesis

source_name: Xenopus embryos treated with TTNPB

source_name: Xenopus embryos treated with AGN193109


title: BMP target genes_set2

description:
Xenopus animal cap cells have properties that closely mimic embryonic stem cells (ES cells).  The cells in the animal cap can be programmed to differentiate into many tissues in vitro by simply treating the tissue with purified growth factors.  Animal cap explants were removed from early gastrula stage (stage 8-9) Xenopus embryos, and the explants were subsequently divided into two populations.  Because intact animal cap explants secrete endogenous BMPs, dissociation of the animal caps into single cells is necessary before treatment with BMP-2.  By first dispersing the explant cells in Ca2+ free saline solution, the endogenous BMPs are diluted and the cellular response becomes dependent on the amount of exogenously added BMPs.  The two populations of dissociated animal cap cells were then bathed in medium containing the protein synthesis inhibitor, cycloheximide (CHX) plus BMP2 protein, or cycloheximide only.  By doing so, I am able to induce target genes that do not require any additional protein synthesis; therefore labeled “direct” target genes.  To ensure that cells remained dissociated for the duration of the treatment, the medium was disturbed every 15 minutes by vigorous pipetting.
During the early gastrula stages (stage 10.5-11), total RNA samples were extracted from the cultured animal cap explants.  From the resulting RNA, cDNA was synthesized via reverse-transcription reactions, incorporating the fluorescently labeled nucleotides Cy3 control (green) and Cy5 experimental (red).  The resulting cDNA was used as probes in hybridization experiments on an arrayed cDNA library.    	The hybridized slide was visualized with the GenePix scanner and the signal intensities of hybridized Cy-3 or Cy-5 labeled probes was calculated.  Results, in triplicate, were compiled to form a list of genes upregulated upon the growth factor treatment along with statistical p-values.

Keywords = Xenopus
Keywords = BMP
Keywords = microarray
Keywords = development
Keywords = target genes

source_name: BMP-treated cells

source_name: control cells


title: Tissue slide12

source_name: Pig 441 small intestine

source_name: Pig 441 muscle


title: Tissue slide7

source_name: Pig 407 Muscle

source_name: Pig 407 small intestine


title: RA responsive genes 2


title: Tissue slide10

source_name: Pig 441 lung

source_name: Pig 441 liver


title: Tissue slide13

source_name: Pig 411 Lung

source_name: Pig 411 small intestine


title: Comparing between control and neural induced ectodermal cell, set1_sample1

source_name: Ectodermal cells those were converted to neuroectodermal cells by inhibition of BMP signalig

source_name: Control ectodermal cell


title: Tissue Slide3

source_name: Pig 436 muscle

source_name: Pig 436 lung


title: Tissue slide1

description:
Total RNA from different porcine tissues, lung, muscle (ham), liver, and small intestine (duodenum; approximately 5 cm from stomach), were isolated by RNeasy kit and treated by DNaseI. For generating amino-allyl-dUTP labeled single strand cDNA, 30 ug RNA from the four tissue samples were reverse transcribed for 2-3 h at 42C in the presence of 6 ug random and 1 ug oligo-dT(18) primers (Invitrogen/Life Technologies), 1 X first strand buffer, 10mM DTT, dNTP (25 mM dCTP, 25 mM dGTP, 25 mM dATP, 15 mM dTTP, 10 mM aa-dUTP), and 400 units of Superscript II reverse transcriptase (Invitrogen/Life Technologies).  RNA was hydrolyzed with 1M NaOH, and then neutralized with 1M HEPES (pH 7.0). First-strand cDNA was precipitated, air dried and re-suspended with 4 ul 0.1M Na2CO3 buffer (pH 9.0), mixed with either Cy3 or Cy5 NHS-ester (Amersham Pharmacia), and incubated 1 hr at room temperature. Cy3 and Cy5 labeled cDNA were purified on QIAquick PCR purification columns (Qiagen, Valencia, CA) according to manufacturer’s instructions, except that QIAquick wash buffer was replaced by phosphate buffer.  Eluted cDNA were precipitated by ETOH and hybridized to the Qiagen-NRSP-8 porcine oligonucleotide microarray at 42C for 12-16 hr in hybridization solution containing 50% formamide, 5 X SSC, 0.1% SDS and 0.2 ug/ul sheared salmon sperm DNA.  After hybridization, microarray slides were washed by 2 X SSC, 0.1% SDS for 5 min at 42C, 0.1 X SSC for 1 min at room temperature for four times, and 0.01 X SSC for 10 sec at room temperature, and dried by centrifugation.Slides were scanned at 10 um resolution using a ScanArray 5000 scanner under conditions to limit saturation to <1% and were saved as TIFF images. The intensities of spots on each image were quantified by ImaGene 5.1 software, and data were saved as .txt files for further analyses.
Keywords = pig
Keywords = microarray
Keywords = tissue
Keywords = gene expression

source_name: Pig 436 Lung

source_name: Pig 436 Small Intestine


title: Tissue slide6

source_name: Pig 407 liver

source_name: Pig 407 muscle


title: Tissue sldie11


title: Tissue slide2

source_name: Pig 436 liver

source_name: pig 436 Muscle


title: Tissue slide8

source_name: Pig 407 lung


title: Tissue Slide4

source_name: Pig 436 small intestine


title: BMP target genes_set1

source_name: BMP-treated cells

source_name: control cells


title: Expression from human TK6 cells treated with 1M N-OH-AABP vs wild type #2

status: Public on Mar 02 2005

city: Seattle

first: Wen

last: Luo

state: WA

address: 1800 East Lake

department: Human Biology

institute: Fred Hutchinson Cancer Research Center

description:
Cell culture

Human lymphoblastoid line TK6 was derived from the parental human lymphoblastoid line HH4 and heterozygous for thymidine kinase (TK), an enzyme which phosphorylates thymidine and its toxic analogs in an ATP-dependent reaction. The cells were grown in spinner flasks to allow for gentle stirring.  Cells were maintained in exponential growth by daily dilution in RPMI 1640 medium supplemented with 5% donor horse serum and maintained in 37oC incubators with a 5% CO2 atmosphere.  Cell counts were taken daily using a Coulter Counter, and cultures were diluted to 4~5´105 cells/mL.  Detailed growth records were maintained and used to ascertain any effects of the treatment on growth rate.

Chemical treatment, mutagenesis and survival measurements

N-OH-AABP and BPDE stock solutions were prepared in anhydrous DMSO (99.997% purity) and used immediately. The final DMSO concentration in the culture was less than 0.1%.

Prior to mutagen treatment, the background HPRT mutant fraction was reduced. TK6 cells were first “CHAT” treated for three days, followed by two days recovery period in “THC”.  Cells were then treated with various concentrations of test chemicals such as BPDE or N-OH-AAB for up to 27 hours. Duplicate cultures were completed at each condition. Untreated cells were used as negative controls. At the end of the treatment, cells were sampled for measurement of the mutant fractions, DNA adducts level and gene _expression. At 1, 9 or 27 hours after the treatment, cells were sampled for mutant fraction.  At sampling, a 100 ml aliquot from each culture was centrifuged, and cells were resuspended in fresh RPMI medium to remove test chemicals. After cells had been growing exponentially for 7 days to allow for phenotypic _expression, cultures were plated to determine mutant fractions. To determine mutant fraction at HPRT or TK locus, cells were plated in the presence and absence of 6-thioguanine (6TG) or triflurothymidine (F3TdR), respectively. The mutant fraction is the ratio of the colony-forming efficiency under selective conditions to that of efficiency in the absence of selective conditions. Survival of treated cultures was calculated from back extrapolation of growth curves by comparison of the growth of treated cultures to control cultures. For analysis of gene _expression and DNA adducts, two 100 ml aliquots of cells (about 5´105 cells/mL) were removed from each culture at 1, 9 or 27 hours after the treatment. Cells were collected by centrifugation, immediately frozen down in liquid nitrogen, and stored at -80oC.

DNA microarrays

Human cDNA arrays were generated in the Genomics Facility at Fred Hutchinson Cancer Research Center. Microarrays were constructed employing the first 17,569 clones from the sequence-verified ResGen Human Unigene Set comprising 31,105 clones (Research Genetics, Huntsville, AL - now Invitrogen). Although each clone was originally representative of a unique UniGene Cluster, members of these clusters have changed over time. Our review of the annotations for 521 clones indicated that redundancy of probes was approximately less than 2.5% (12/521). Clones are representative of over 20 different human tissues/organs.  The average length of the probe was 1200 ± 364 bases, with the minimum probe length being 251 bases and the maximum probe length being 2000+ bases.

For array construction, each clone insert was individually amplified using PCR. Each PCR product was then verified on the basis of size using gel electrophoresis, and purified using the Millipore Multiscreen-PCR filtration system. In some instances, re-sequencing was performed to verify the identity of clones. Purified PCR products in 3X SSC (450 mM sodium chloride and 45 mM sodium citrate, pH 7.0) were each mechanically “spotted” onto poly-lysine coated microscope slides using an OmniGrid high-precision robotic gridder (GeneMachines, San Carlo, CA).  Each clone was represented once per array.

Preparation and hybridization of cDNA probes labeled with Cy3 or Cy5

RNA was extracted using Qiagen RNeasyMaxi Kits according to the manufacturer’s instructions (Qiagen, Valencia, CA), followed by a quality test using Agilent BioAnalyzerächips. Synthesis of the labeled first strand cDNA was conducted using InVitrogen Superscript II reverse transcript system with starting material of 30 µg total RNA plus oligo-dT. The amino-allyl labeled dUTP was added to the reaction to generate amino-allyl labeled second strand cDNA. Following the hydrolysis reaction, single-stranded cDNA probes were purified using a Microcon–30 concentrator. Probe mixtures were evaporated in a vacuum centrifuge, and the cDNA pellet was resuspended in 4.5 µL of water. The dye coupling reactions were performed by mixing the cDNA samples with Cy3 or Cy5 dyes and incubating for 1 hour in the dark. After the reactions were quenched with hydroxylamine, Cy3 and Cy5 samples were combined for hybridization. To remove unincorporated/quenched Cy dye, a Qia-quick PCR purification kit (Qiagen) was used. The reactions were purified with Qiaquik PCR purification kits to remove the unincorporated/quenched dyes. The mixture of Cy3 and Cy5 labeled samples was dried down in a vacuum centrifuge and then brought to a volume of 20 µL with water. Millipore 0.45 µM spin columns were used to clean up after adding 20X SSC and Poly A to the mixture. Finally, the samples were stored at –20o C until ready for hybridization.

The labeled cDNAs were co-hybridized to cDNA microarrays in triplicate, including one dye swap. Slides were scanned on the GenePix 4000A Microarray Scanner at the optimal wavelength for the Cy3 and Cy5 using lasers. The fluorescence signals of each spot on the slide were analyzed and extracted with GenePix software to generate .gpr file, which was further formatted to plain .txt file for further data analysis. All the data mining steps including transformation, cleaning-up, normalization and hypothesis tests, were performed using the SAS statistical software.

organism: Homo sapiens

source_name: wild type K6 cells, 0 h, cy5

source_name: TK6 cells treated with 1 uM N-OH-AABP, 27h, cy3


title: TK6 cells treated with 10 uM N-OH-AABP, 27h, cy3


title: Expression from human TK6 cells treated with 0.5uM N-OH-AABP vs wild type #3

source_name: TK6 cells treated with 0.5 UM N-OH-AABP, 27h, cy5


title: Comparing between control and neural induced ectodermal cell, set1_sample2

source_name: Source:Ectodermal cells those were converted to neuroectodermal cells by inhibition of BMP signalig


title: Expression from human TK6 cells treated with 17 nM BPDE vs wild type #2

source_name: TK6 cells treated with 17 nM BPDE , 1h, cy3


title: Expression from human TK6 cells treated with 1M N-OH-AABP vs wild type #3

source_name: TK6 cells treated with 1 UM N-OH-AABP, 27h, cy5


title: Expression from human TK6 cells treated with 10 uM N-OH-AABP vs wild type #3

source_name: TK6 cells treated with 10 UM N-OH-AABP, 27h, cy5


title: Comparing between control and neural induced ectodermal cell, set2_sample1

source_name: Ectodermal cells those were converted to neuroectodermal cells by inhibition of BMP signalig

source_name: Control ectodermal cells


title: Tissue slide9

source_name: Pig 441 muscle

source_name: Pig 441 lung


title: Expression from human TK6 cells treated with 120 nM BPDE vs wild type

source_name: TK6 cells treated with 120 nM BPDE, 1h, cy5

source_name: wild type K6 cells, 0 h, cy3


title: Tissue silde14

source_name: Pig 411 liver

source_name: Pig 411 muscle


title: Expression from human TK6 cells treated with 1 uM N-OH-AABP vs wild type

description:
Material

Benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide(±)(anti) (BPDE) and N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) were purchased from National Cancer Institute Chemical Carcinogen Reference Standard Repository (Kansas City, MO).

Cell culture

Human lymphoblastoid line TK6 was derived from the parental human lymphoblastoid line HH4 and heterozygous for thymidine kinase (TK), an enzyme which phosphorylates thymidine and its toxic analogs in an ATP-dependent reaction. The cells were grown in spinner flasks to allow for gentle stirring.  Cells were maintained in exponential growth by daily dilution in RPMI 1640 medium supplemented with 5% donor horse serum and maintained in 37oC incubators with a 5% CO2 atmosphere.  Cell counts were taken daily using a Coulter Counter, and cultures were diluted to 4~5´105 cells/mL.  Detailed growth records were maintained and used to ascertain any effects of the treatment on growth rate.

Chemical treatment, mutagenesis and survival measurements

N-OH-AABP and BPDE stock solutions were prepared in anhydrous DMSO (99.997% purity) and used immediately. The final DMSO concentration in the culture was less than 0.1%.

Prior to mutagen treatment, the background HPRT mutant fraction was reduced. TK6 cells were first “CHAT” treated for three days, followed by two days recovery period in “THC”.  Cells were then treated with various concentrations of test chemicals such as BPDE or N-OH-AAB for up to 27 hours. Duplicate cultures were completed at each condition. Untreated cells were used as negative controls. At the end of the treatment, cells were sampled for measurement of the mutant fractions, DNA adducts level and gene _expression. At 1, 9 or 27 hours after the treatment, cells were sampled for mutant fraction.  At sampling, a 100 ml aliquot from each culture was centrifuged, and cells were resuspended in fresh RPMI medium to remove test chemicals. After cells had been growing exponentially for 7 days to allow for phenotypic _expression, cultures were plated to determine mutant fractions. To determine mutant fraction at HPRT or TK locus, cells were plated in the presence and absence of 6-thioguanine (6TG) or triflurothymidine (F3TdR), respectively. The mutant fraction is the ratio of the colony-forming efficiency under selective conditions to that of efficiency in the absence of selective conditions. Survival of treated cultures was calculated from back extrapolation of growth curves by comparison of the growth of treated cultures to control cultures. For analysis of gene _expression and DNA adducts, two 100 ml aliquots of cells (about 5´105 cells/mL) were removed from each culture at 1, 9 or 27 hours after the treatment. Cells were collected by centrifugation, immediately frozen down in liquid nitrogen, and stored at -80oC.

DNA microarrays

Human cDNA arrays were generated in the Genomics Facility at Fred Hutchinson Cancer Research Center. Microarrays were constructed employing the first 17,569 clones from the sequence-verified ResGen Human Unigene Set comprising 31,105 clones (Research Genetics, Huntsville, AL - now Invitrogen). Although each clone was originally representative of a unique UniGene Cluster, members of these clusters have changed over time. Our review of the annotations for 521 clones indicated that redundancy of probes was approximately less than 2.5% (12/521). Clones are representative of over 20 different human tissues/organs.  The average length of the probe was 1200 ± 364 bases, with the minimum probe length being 251 bases and the maximum probe length being 2000+ bases.

For array construction, each clone insert was individually amplified using PCR. Each PCR product was then verified on the basis of size using gel electrophoresis, and purified using the Millipore Multiscreen-PCR filtration system. In some instances, re-sequencing was performed to verify the identity of clones. Purified PCR products in 3X SSC (450 mM sodium chloride and 45 mM sodium citrate, pH 7.0) were each mechanically “spotted” onto poly-lysine coated microscope slides using an OmniGrid high-precision robotic gridder (GeneMachines, San Carlo, CA).  Each clone was represented once per array.

Preparation and hybridization of cDNA probes labeled with Cy3 or Cy5

RNA was extracted using Qiagen RNeasyMaxi Kits according to the manufacturer’s instructions (Qiagen, Valencia, CA), followed by a quality test using Agilent BioAnalyzerächips. Synthesis of the labeled first strand cDNA was conducted using InVitrogen Superscript II reverse transcript system with starting material of 30 µg total RNA plus oligo-dT. The amino-allyl labeled dUTP was added to the reaction to generate amino-allyl labeled second strand cDNA. Following the hydrolysis reaction, single-stranded cDNA probes were purified using a Microcon–30 concentrator. Probe mixtures were evaporated in a vacuum centrifuge, and the cDNA pellet was resuspended in 4.5 µL of water. The dye coupling reactions were performed by mixing the cDNA samples with Cy3 or Cy5 dyes and incubating for 1 hour in the dark. After the reactions were quenched with hydroxylamine, Cy3 and Cy5 samples were combined for hybridization. To remove unincorporated/quenched Cy dye, a Qia-quick PCR purification kit (Qiagen) was used. The reactions were purified with Qiaquik PCR purification kits to remove the unincorporated/quenched dyes. The mixture of Cy3 and Cy5 labeled samples was dried down in a vacuum centrifuge and then brought to a volume of 20 µL with water. Millipore 0.45 µM spin columns were used to clean up after adding 20X SSC and Poly A to the mixture. Finally, the samples were stored at –20o C until ready for hybridization.

The labeled cDNAs were co-hybridized to cDNA microarrays in triplicate, including one dye swap. Slides were scanned on the GenePix 4000A Microarray Scanner at the optimal wavelength for the Cy3 and Cy5 using lasers. The fluorescence signals of each spot on the slide were analyzed and extracted with GenePix software to generate .gpr file, which was further formatted to plain .txt file for further data analysis. All the data mining steps including transformation, cleaning-up, normalization and hypothesis tests, were performed using the SAS statistical software.


title: RA responsive genes

source_name: Xenopus embryos treated with TTNPB

source_name: Xenopus embryos treated with AGN193109


title: Expression from human TK6 cells treated with 17 nM BPDE vs wild type #3

source_name: TK6 cells treated with 17 nM BPDE, 1h, cy5

source_name: wild type K6 cells, 0 h, cy5


title: Expression from wild type human TK6 cells

description:
Material

Benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide(±)(anti) (BPDE) and N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) were purchased from National Cancer Institute Chemical Carcinogen Reference Standard Repository (Kansas City, MO).

Cell culture

Human lymphoblastoid line TK6 was derived from the parental human lymphoblastoid line HH4 and heterozygous for thymidine kinase (TK), an enzyme which phosphorylates thymidine and its toxic analogs in an ATP-dependent reaction. The cells were grown in spinner flasks to allow for gentle stirring.  Cells were maintained in exponential growth by daily dilution in RPMI 1640 medium supplemented with 5% donor horse serum and maintained in 37oC incubators with a 5% CO2 atmosphere.  Cell counts were taken daily using a Coulter Counter, and cultures were diluted to 4~5´105 cells/mL.  Detailed growth records were maintained and used to ascertain any effects of the treatment on growth rate.

Chemical treatment, mutagenesis and survival measurements

N-OH-AABP and BPDE stock solutions were prepared in anhydrous DMSO (99.997% purity) and used immediately. The final DMSO concentration in the culture was less than 0.1%.

Prior to mutagen treatment, the background HPRT mutant fraction was reduced. TK6 cells were first “CHAT” treated for three days, followed by two days recovery period in “THC”.  Cells were then treated with various concentrations of test chemicals such as BPDE or N-OH-AAB for up to 27 hours. Duplicate cultures were completed at each condition. Untreated cells were used as negative controls. At the end of the treatment, cells were sampled for measurement of the mutant fractions, DNA adducts level and gene _expression. At 1, 9 or 27 hours after the treatment, cells were sampled for mutant fraction.  At sampling, a 100 ml aliquot from each culture was centrifuged, and cells were resuspended in fresh RPMI medium to remove test chemicals. After cells had been growing exponentially for 7 days to allow for phenotypic _expression, cultures were plated to determine mutant fractions. To determine mutant fraction at HPRT or TK locus, cells were plated in the presence and absence of 6-thioguanine (6TG) or triflurothymidine (F3TdR), respectively. The mutant fraction is the ratio of the colony-forming efficiency under selective conditions to that of efficiency in the absence of selective conditions. Survival of treated cultures was calculated from back extrapolation of growth curves by comparison of the growth of treated cultures to control cultures. For analysis of gene _expression and DNA adducts, two 100 ml aliquots of cells (about 5´105 cells/mL) were removed from each culture at 1, 9 or 27 hours after the treatment. Cells were collected by centrifugation, immediately frozen down in liquid nitrogen, and stored at -80oC.

DNA microarrays

Human cDNA arrays were generated in the Genomics Facility at Fred Hutchinson Cancer Research Center. Microarrays were constructed employing the first 17,569 clones from the sequence-verified ResGen Human Unigene Set comprising 31,105 clones (Research Genetics, Huntsville, AL - now Invitrogen). Although each clone was originally representative of a unique UniGene Cluster, members of these clusters have changed over time. Our review of the annotations for 521 clones indicated that redundancy of probes was approximately less than 2.5% (12/521). Clones are representative of over 20 different human tissues/organs.  The average length of the probe was 1200 ± 364 bases, with the minimum probe length being 251 bases and the maximum probe length being 2000+ bases.

For array construction, each clone insert was individually amplified using PCR. Each PCR product was then verified on the basis of size using gel electrophoresis, and purified using the Millipore Multiscreen-PCR filtration system. In some instances, re-sequencing was performed to verify the identity of clones. Purified PCR products in 3X SSC (450 mM sodium chloride and 45 mM sodium citrate, pH 7.0) were each mechanically “spotted” onto poly-lysine coated microscope slides using an OmniGrid high-precision robotic gridder (GeneMachines, San Carlo, CA).  Each clone was represented once per array.

Preparation and hybridization of cDNA probes labeled with Cy3 or Cy5

RNA was extracted using Qiagen RNeasyMaxi Kits according to the manufacturer’s instructions (Qiagen, Valencia, CA), followed by a quality test using Agilent BioAnalyzerächips. Synthesis of the labeled first strand cDNA was conducted using InVitrogen Superscript II reverse transcript system with starting material of 30 µg total RNA plus oligo-dT. The amino-allyl labeled dUTP was added to the reaction to generate amino-allyl labeled second strand cDNA. Following the hydrolysis reaction, single-stranded cDNA probes were purified using a Microcon–30 concentrator. Probe mixtures were evaporated in a vacuum centrifuge, and the cDNA pellet was resuspended in 4.5 µL of water. The dye coupling reactions were performed by mixing the cDNA samples with Cy3 or Cy5 dyes and incubating for 1 hour in the dark. After the reactions were quenched with hydroxylamine, Cy3 and Cy5 samples were combined for hybridization. To remove unincorporated/quenched Cy dye, a Qia-quick PCR purification kit (Qiagen) was used. The reactions were purified with Qiaquik PCR purification kits to remove the unincorporated/quenched dyes. The mixture of Cy3 and Cy5 labeled samples was dried down in a vacuum centrifuge and then brought to a volume of 20 µL with water. Millipore 0.45 µM spin columns were used to clean up after adding 20X SSC and Poly A to the mixture. Finally, the samples were stored at –20o C until ready for hybridization.

The labeled cDNAs were co-hybridized to cDNA microarrays in triplicate, including one dye swap. Slides were scanned on the GenePix 4000A Microarray Scanner at the optimal wavelength for the Cy3 and Cy5 using lasers. The fluorescence signals of each spot on the slide were analyzed and extracted with GenePix software to generate .gpr file, which was further formatted to plain .txt file for further data analysis. All the data mining steps including transformation, cleaning-up, normalization and hypothesis tests, were performed using the SAS statistical software.
Data Analysis

The mean log2 ratios of fluorescence intensities relative to untreated controls for each gene and each treatment were clustered using the J-Express Software (University of Bergen, Norway) unsupervised algorithm. Briefly, the fluorescence intensity of each spot was calculated using local median background subtraction. The relative fluorescent units (RFUs) were then normalized to the median signal (Cy3 and Cy5) for that slide. If both Cy3 and Cy5 signals were under the signal intensity of negative controls, genes were considered as not being expressed in either sample. The normalized microarray data were then analyzed at using two approaches, visualization and statistical analysis. The first of these involved a comparison of the normalized fluorescence intensity ratios, and was used only for the purpose of visualizing the data.  In this approach, the fold change in gene _expression for each array feature was determined as a ratio of normalized fluorescence intensities relative to the reference sample (i.e, RFU treated/RFU untreated). We then selected a value of 2.0 in fold change in gene _expression as the arbitrary cut-off for inclusion of data in clustering algorithms. The log2 gene _expression ratios for all genes showing a 2-fold or greater change in gene _expression at any concentration were used in unsupervised cluster analysis to generate the dendogram shown in Figure 3.  The data were also analyzed by multi-dimensional scaling using Self Organization Map techniques. The arbitrary 2-fold cut-off was selected because it is the most frequently used value for preliminary comparison of _expression profiles. As illustrate

source_name: wild type TK6 cells, 0 h, cy5


title: Expression from human TK6 cells treated with 34 nM BPDE vs wild type #3

description:
Cell cultureCell culture

Human lymphoblastoid line TK6 was derived from the parental human lymphoblastoid line HH4 and heterozygous for thymidine kinase (TK), an enzyme which phosphorylates thymidine and its toxic analogs in an ATP-dependent reaction. The cells were grown in spinner flasks to allow for gentle stirring.  Cells were maintained in exponential growth by daily dilution in RPMI 1640 medium supplemented with 5% donor horse serum and maintained in 37oC incubators with a 5% CO2 atmosphere.  Cell counts were taken daily using a Coulter Counter, and cultures were diluted to 4~5´105 cells/mL.  Detailed growth records were maintained and used to ascertain any effects of the treatment on growth rate.

Chemical treatment, mutagenesis and survival measurements

N-OH-AABP and BPDE stock solutions were prepared in anhydrous DMSO (99.997% purity) and used immediately. The final DMSO concentration in the culture was less than 0.1%.

Prior to mutagen treatment, the background HPRT mutant fraction was reduced. TK6 cells were first “CHAT” treated for three days, followed by two days recovery period in “THC”.  Cells were then treated with various concentrations of test chemicals such as BPDE or N-OH-AAB for up to 27 hours. Duplicate cultures were completed at each condition. Untreated cells were used as negative controls. At the end of the treatment, cells were sampled for measurement of the mutant fractions, DNA adducts level and gene _expression. At 1, 9 or 27 hours after the treatment, cells were sampled for mutant fraction.  At sampling, a 100 ml aliquot from each culture was centrifuged, and cells were resuspended in fresh RPMI medium to remove test chemicals. After cells had been growing exponentially for 7 days to allow for phenotypic _expression, cultures were plated to determine mutant fractions. To determine mutant fraction at HPRT or TK locus, cells were plated in the presence and absence of 6-thioguanine (6TG) or triflurothymidine (F3TdR), respectively. The mutant fraction is the ratio of the colony-forming efficiency under selective conditions to that of efficiency in the absence of selective conditions. Survival of treated cultures was calculated from back extrapolation of growth curves by comparison of the growth of treated cultures to control cultures. For analysis of gene _expression and DNA adducts, two 100 ml aliquots of cells (about 5´105 cells/mL) were removed from each culture at 1, 9 or 27 hours after the treatment. Cells were collected by centrifugation, immediately frozen down in liquid nitrogen, and stored at -80oC.

DNA microarrays

Human cDNA arrays were generated in the Genomics Facility at Fred Hutchinson Cancer Research Center. Microarrays were constructed employing the first 17,569 clones from the sequence-verified ResGen Human Unigene Set comprising 31,105 clones (Research Genetics, Huntsville, AL - now Invitrogen). Although each clone was originally representative of a unique UniGene Cluster, members of these clusters have changed over time. Our review of the annotations for 521 clones indicated that redundancy of probes was approximately less than 2.5% (12/521). Clones are representative of over 20 different human tissues/organs.  The average length of the probe was 1200 ± 364 bases, with the minimum probe length being 251 bases and the maximum probe length being 2000+ bases.

For array construction, each clone insert was individually amplified using PCR. Each PCR product was then verified on the basis of size using gel electrophoresis, and purified using the Millipore Multiscreen-PCR filtration system. In some instances, re-sequencing was performed to verify the identity of clones. Purified PCR products in 3X SSC (450 mM sodium chloride and 45 mM sodium citrate, pH 7.0) were each mechanically “spotted” onto poly-lysine coated microscope slides using an OmniGrid high-precision robotic gridder (GeneMachines, San Carlo, CA).  Each clone was represented once per array.

Preparation and hybridization of cDNA probes labeled with Cy3 or Cy5

RNA was extracted using Qiagen RNeasyMaxi Kits according to the manufacturer’s instructions (Qiagen, Valencia, CA), followed by a quality test using Agilent BioAnalyzerächips. Synthesis of the labeled first strand cDNA was conducted using InVitrogen Superscript II reverse transcript system with starting material of 30 µg total RNA plus oligo-dT. The amino-allyl labeled dUTP was added to the reaction to generate amino-allyl labeled second strand cDNA. Following the hydrolysis reaction, single-stranded cDNA probes were purified using a Microcon–30 concentrator. Probe mixtures were evaporated in a vacuum centrifuge, and the cDNA pellet was resuspended in 4.5 µL of water. The dye coupling reactions were performed by mixing the cDNA samples with Cy3 or Cy5 dyes and incubating for 1 hour in the dark. After the reactions were quenched with hydroxylamine, Cy3 and Cy5 samples were combined for hybridization. To remove unincorporated/quenched Cy dye, a Qia-quick PCR purification kit (Qiagen) was used. The reactions were purified with Qiaquik PCR purification kits to remove the unincorporated/quenched dyes. The mixture of Cy3 and Cy5 labeled samples was dried down in a vacuum centrifuge and then brought to a volume of 20 µL with water. Millipore 0.45 µM spin columns were used to clean up after adding 20X SSC and Poly A to the mixture. Finally, the samples were stored at –20o C until ready for hybridization.

The labeled cDNAs were co-hybridized to cDNA microarrays in triplicate, including one dye swap. Slides were scanned on the GenePix 4000A Microarray Scanner at the optimal wavelength for the Cy3 and Cy5 using lasers. The fluorescence signals of each spot on the slide were analyzed and extracted with GenePix software to generate .gpr file, which was further formatted to plain .txt file for further data analysis. All the data mining steps including transformation, cleaning-up, normalization and hypothesis tests, were performed using the SAS statistical software.

source_name: TK6 cells treated with 34 nM BPDE, 1h, cy5


title: Expression from human TK6 cells treated with 34 nM BPDE vs wild type

description:
Benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide(±)(anti) (BPDE) and N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) were purchased from National Cancer Institute Chemical Carcinogen Reference Standard Repository (Kansas City, MO).

Cell culture

Human lymphoblastoid line TK6 was derived from the parental human lymphoblastoid line HH4 and heterozygous for thymidine kinase (TK), an enzyme which phosphorylates thymidine and its toxic analogs in an ATP-dependent reaction. The cells were grown in spinner flasks to allow for gentle stirring.  Cells were maintained in exponential growth by daily dilution in RPMI 1640 medium supplemented with 5% donor horse serum and maintained in 37oC incubators with a 5% CO2 atmosphere.  Cell counts were taken daily using a Coulter Counter, and cultures were diluted to 4~5´105 cells/mL.  Detailed growth records were maintained and used to ascertain any effects of the treatment on growth rate.

Chemical treatment, mutagenesis and survival measurements

N-OH-AABP and BPDE stock solutions were prepared in anhydrous DMSO (99.997% purity) and used immediately. The final DMSO concentration in the culture was less than 0.1%.

Prior to mutagen treatment, the background HPRT mutant fraction was reduced. TK6 cells were first “CHAT” treated for three days, followed by two days recovery period in “THC”.  Cells were then treated with various concentrations of test chemicals such as BPDE or N-OH-AAB for up to 27 hours. Duplicate cultures were completed at each condition. Untreated cells were used as negative controls. At the end of the treatment, cells were sampled for measurement of the mutant fractions, DNA adducts level and gene _expression. At 1, 9 or 27 hours after the treatment, cells were sampled for mutant fraction.  At sampling, a 100 ml aliquot from each culture was centrifuged, and cells were resuspended in fresh RPMI medium to remove test chemicals. After cells had been growing exponentially for 7 days to allow for phenotypic _expression, cultures were plated to determine mutant fractions. To determine mutant fraction at HPRT or TK locus, cells were plated in the presence and absence of 6-thioguanine (6TG) or triflurothymidine (F3TdR), respectively. The mutant fraction is the ratio of the colony-forming efficiency under selective conditions to that of efficiency in the absence of selective conditions. Survival of treated cultures was calculated from back extrapolation of growth curves by comparison of the growth of treated cultures to control cultures. For analysis of gene _expression and DNA adducts, two 100 ml aliquots of cells (about 5´105 cells/mL) were removed from each culture at 1, 9 or 27 hours after the treatment. Cells were collected by centrifugation, immediately frozen down in liquid nitrogen, and stored at -80oC.

DNA microarrays

Human cDNA arrays were generated in the Genomics Facility at Fred Hutchinson Cancer Research Center. Microarrays were constructed employing the first 17,569 clones from the sequence-verified ResGen Human Unigene Set comprising 31,105 clones (Research Genetics, Huntsville, AL - now Invitrogen). Although each clone was originally representative of a unique UniGene Cluster, members of these clusters have changed over time. Our review of the annotations for 521 clones indicated that redundancy of probes was approximately less than 2.5% (12/521). Clones are representative of over 20 different human tissues/organs.  The average length of the probe was 1200 ± 364 bases, with the minimum probe length being 251 bases and the maximum probe length being 2000+ bases.

For array construction, each clone insert was individually amplified using PCR. Each PCR product was then verified on the basis of size using gel electrophoresis, and purified using the Millipore Multiscreen-PCR filtration system. In some instances, re-sequencing was performed to verify the identity of clones. Purified PCR products in 3X SSC (450 mM sodium chloride and 45 mM sodium citrate, pH 7.0) were each mechanically “spotted” onto poly-lysine coated microscope slides using an OmniGrid high-precision robotic gridder (GeneMachines, San Carlo, CA).  Each clone was represented once per array.

Preparation and hybridization of cDNA probes labeled with Cy3 or Cy5

RNA was extracted using Qiagen RNeasyMaxi Kits according to the manufacturer’s instructions (Qiagen, Valencia, CA), followed by a quality test using Agilent BioAnalyzerächips. Synthesis of the labeled first strand cDNA was conducted using InVitrogen Superscript II reverse transcript system with starting material of 30 µg total RNA plus oligo-dT. The amino-allyl labeled dUTP was added to the reaction to generate amino-allyl labeled second strand cDNA. Following the hydrolysis reaction, single-stranded cDNA probes were purified using a Microcon–30 concentrator. Probe mixtures were evaporated in a vacuum centrifuge, and the cDNA pellet was resuspended in 4.5 µL of water. The dye coupling reactions were performed by mixing the cDNA samples with Cy3 or Cy5 dyes and incubating for 1 hour in the dark. After the reactions were quenched with hydroxylamine, Cy3 and Cy5 samples were combined for hybridization. To remove unincorporated/quenched Cy dye, a Qia-quick PCR purification kit (Qiagen) was used. The reactions were purified with Qiaquik PCR purification kits to remove the unincorporated/quenched dyes. The mixture of Cy3 and Cy5 labeled samples was dried down in a vacuum centrifuge and then brought to a volume of 20 µL with water. Millipore 0.45 µM spin columns were used to clean up after adding 20X SSC and Poly A to the mixture. Finally, the samples were stored at –20o C until ready for hybridization.

The labeled cDNAs were co-hybridized to cDNA microarrays in triplicate, including one dye swap. Slides were scanned on the GenePix 4000A Microarray Scanner at the optimal wavelength for the Cy3 and Cy5 using lasers. The fluorescence signals of each spot on the slide were analyzed and extracted with GenePix software to generate .gpr file, which was further formatted to plain .txt file for further data analysis. All the data mining steps including transformation, cleaning-up, normalization and hypothesis tests, were performed using the SAS statistical software.


title: Expression from wild type human TK6 cells #2


title: Expression from human TK6 cells treated with 120 nM BPDE vs wild type #3


title: Expression from human TK6 cells treated with 0.5uM N-OH-AABP vs wild type #2

description:
Material

Benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide(±)(anti) (BPDE) and N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) were purchased from National Cancer Institute Chemical Carcinogen Reference Standard Repository (Kansas City, MO).

Cell culture

Human lymphoblastoid line TK6 was derived from the parental human lymphoblastoid line HH4 and heterozygous for thymidine kinase (TK), an enzyme which phosphorylates thymidine and its toxic analogs in an ATP-dependent reaction. The cells were grown in spinner flasks to allow for gentle stirring.  Cells were maintained in exponential growth by daily dilution in RPMI 1640 medium supplemented with 5% donor horse serum and maintained in 37oC incubators with a 5% CO2 atmosphere.  Cell counts were taken daily using a Coulter Counter, and cultures were diluted to 4~5´105 cells/mL.  Detailed growth records were maintained and used to ascertain any effects of the treatment on growth rate.

Chemical treatment, mutagenesis and survival measurements
N-OH-AABP and BPDE stock solutions were prepared in anhydrous DMSO (99.997% purity) and used immediately. The final DMSO concentration in the culture was less than 0.1%.

Prior to mutagen treatment, the background HPRT mutant fraction was reduced. TK6 cells were first “CHAT” treated for three days, followed by two days recovery period in “THC”.  Cells were then treated with various concentrations of test chemicals such as BPDE or N-OH-AAB for up to 27 hours. Duplicate cultures were completed at each condition. Untreated cells were used as negative controls. At the end of the treatment, cells were sampled for measurement of the mutant fractions, DNA adducts level and gene expression. At 1, 9 or 27 hours after the treatment, cells were sampled for mutant fraction.  At sampling, a 100 ml aliquot from each culture was centrifuged, and cells were resuspended in fresh RPMI medium to remove test chemicals. After cells had been growing exponentially for 7 days to allow for phenotypic expression, cultures were plated to determine mutant fractions. To determine mutant fraction at HPRT or TK locus, cells were plated in the presence and absence of 6-thioguanine (6TG) or triflurothymidine (F3TdR), respectively. The mutant fraction is the ratio of the colony-forming efficiency under selective conditions to that of efficiency in the absence of selective conditions. Survival of treated cultures was calculated from back extrapolation of growth curves by comparison of the growth of treated cultures to control cultures. For analysis of gene expression and DNA adducts, two 100 ml aliquots of cells (about 5´105 cells/mL) were removed from each culture at 1, 9 or 27 hours after the treatment. Cells were collected by centrifugation, immediately frozen down in liquid nitrogen, and stored at -80oC.

DNA microarrays

Human cDNA arrays were generated in the Genomics Facility at Fred Hutchinson Cancer Research Center. Microarrays were constructed employing the first 17,569 clones from the sequence-verified ResGen Human Unigene Set comprising 31,105 clones (Research Genetics, Huntsville, AL - now Invitrogen). Although each clone was originally representative of a unique UniGene Cluster, members of these clusters have changed over time. Our review of the annotations for 521 clones indicated that redundancy of probes was approximately less than 2.5% (12/521). Clones are representative of over 20 different human tissues/organs.  The average length of the probe was 1200 ± 364 bases, with the minimum probe length being 251 bases and the maximum probe length being 2000+ bases.

For array construction, each clone insert was individually amplified using PCR. Each PCR product was then verified on the basis of size using gel electrophoresis, and purified using the Millipore Multiscreen-PCR filtration system. In some instances, re-sequencing was performed to verify the identity of clones. Purified PCR products in 3X SSC (450 mM sodium chloride and 45 mM sodium citrate, pH 7.0) were each mechanically “spotted” onto poly-lysine coated microscope slides using an OmniGrid high-precision robotic gridder (GeneMachines, San Carlo, CA).  Each clone was represented once per array.

Preparation and hybridization of cDNA probes labeled with Cy3 or Cy5

RNA was extracted using Qiagen RNeasyMaxi Kits according to the manufacturer’s instructions (Qiagen, Valencia, CA), followed by a quality test using Agilent BioAnalyzerächips. Synthesis of the labeled first strand cDNA was conducted using InVitrogen Superscript II reverse transcript system with starting material of 30 µg total RNA plus oligo-dT. The amino-allyl labeled dUTP was added to the reaction to generate amino-allyl labeled second strand cDNA. Following the hydrolysis reaction, single-stranded cDNA probes were purified using a Microcon–30 concentrator. Probe mixtures were evaporated in a vacuum centrifuge, and the cDNA pellet was resuspended in 4.5 µL of water. The dye coupling reactions were performed by mixing the cDNA samples with Cy3 or Cy5 dyes and incubating for 1 hour in the dark. After the reactions were quenched with hydroxylamine, Cy3 and Cy5 samples were combined for hybridization. To remove unincorporated/quenched Cy dye, a Qia-quick PCR purification kit (Qiagen) was used. The reactions were purified with Qiaquik PCR purification kits to remove the unincorporated/quenched dyes. The mixture of Cy3 and Cy5 labeled samples was dried down in a vacuum centrifuge and then brought to a volume of 20 µL with water. Millipore 0.45 µM spin columns were used to clean up after adding 20X SSC and Poly A to the mixture. Finally, the samples were stored at –20o C until ready for hybridization.

The labeled cDNAs were co-hybridized to cDNA microarrays in triplicate, including one dye swap. Slides were scanned on the GenePix 4000A Microarray Scanner at the optimal wavelength for the Cy3 and Cy5 using lasers. The fluorescence signals of each spot on the slide were analyzed and extracted with GenePix software to generate .gpr file, which was further formatted to plain .txt file for further data analysis. All the data mining steps including transformation, cleaning-up, normalization and hypothesis tests, were performed using the SAS statistical software.

Data Analysis
The mean log2 ratios of fluorescence intensities relative to untreated controls for each gene and each treatment were clustered using the J-Express Software (University of Bergen, Norway) unsupervised algorithm. Briefly, the fluorescence intensity of each spot was calculated using local median background subtraction. The relative fluorescent units (RFUs) were then normalized to the median signal (Cy3 and Cy5) for that slide. If both Cy3 and Cy5 signals were under the signal intensity of negative controls, genes were considered as not being expressed in either sample. The normalized microarray data were then analyzed at using two approaches, visualization and statistical analysis. The first of these involved a comparison of the normalized fluorescence intensity ratios, and was used only for the purpose of visualizing the data.  In this approach, the fold change in gene expression for each array feature was determined as a ratio of normalized fluorescence intensities relative to the reference sample (i.e, RFU treated/RFU untreated). We then selected a value of 2.0 in fold change in gene expression as the arbitrary cut-off for inclusion of data in clustering algorithms. The log2 gene expression ratios for all genes showing a 2-fold or greater change in gene expression at any concentration were used in unsupervised cluster analysis to generate the dendogram shown in Figure 3.  The data were also analyzed by multi-dimensional scaling using Self Organization Map techniques. The arbitrary 2-fold cut-off was selected because it is the most frequently used value for preliminary comparison of expression profiles. As illustrated in Figu

source_name: TK6 cells treated with 0.5 uM N-OH-AABP, 27h, cy3


title: Expression from human TK6 cells treated with 10 uM N-OH-AABP vs wild type


title: Expression from human TK6 cells treated with 120 nM BPDE vs wild type #2

source_name: TK6 cells treated with 120 nM BPDE , 1h, cy3


title: Tissue slide5

source_name: Pig 407 lung

source_name: Pig 407 liver


title: Expression from human TK6 cells treated with 17 nM BPDE vs wild type


title: Expression from human TK6 cells treated with 34 nM BPDE vs wild type #2

source_name: TK6 cells treated with 34 nM BPDE , 1h, cy3


title: yellow experiment 4

status: Public on Mar 01 2005

city: Madrid

first: Javier

last: Arroyo

state: Madrid

address: Plaza Ramon y Cajal S/N

department: Microbiologia II

country: Spain

institute: Facultad de Farmacia (UCM)

description: S. cerevisiae was grown on YEPD. For yellow experiments, yeast cells were grown overnight at 24 °C to an optical density 0.8 - 1 (A600). The culture was refreshed to 0.2 O.D and grown at 24 °C for 6h.  At this time cells were collected. Experiment was carried out hybridising the cDNA from the same RNA (from this culture) with both Cy3 and Cy5

source_name: BY4741 Strain (Control mRNA: no treatment)