title: HMVEC Control of Endostatin

description:
Control RNA was extracted from untreated HMVEC. Total RNA was isolated using TRIZOL solution (Invitrogen, Carlsbad, CA). Briefly, after treatment, cells were washed twice in PBS and incubated with TRIZOL for 5 minutes at room temperature. RNA was extracted with 200 ul chloroform per ml TRIZOL and precipitated with 100% isopropanol at -20ºC. The RNA pellet was resuspended in DEPC-treated water and stored at –80ºC until further use. Human cDNA microarrays (Research Genetics/Invitrogen, Carlsbad, CA) consisted of nylon membranes containing 4,132 named genes EST’s (GF211). The array also contained 192 spots of total genomic DNA that served as reference points for the image analysis software Pathways 2.0 (Research Genetics/Invitrogen, Carlsbad, CA) for normalization purposes and to verify the homogeneity of the hybridization. cDNA probes were made from 5 micro-g total RNA with α[33P]-dATP (Amersham Biosciences, Piscataway, NJ) by oligo dT-primed polymerization using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). Probes were purified by gel chromatography (BioSpin 6; BioRad Laboratories, Hercules, CA), boiled for about 3 min and allowed to cool to room temperature. The microarrays were pre-hybridized for no less than 2 hr and then hybridized overnight at 42 oC. Filters were washed with 2xSSC and 1 % SDS for 15 min at 50 oC, and then with 0.5xSSC and 1% SDS twice for a total of 30 min at room temperature. Washed filters were then exposed to phosphor imager screens, which were scanned by a Molecular Dynamics Storm Imager (Packard Instruments, Meriden, CT) at 600 dpi resolution. Scanned files containing the microarray were analyzed with Pathways software (Invitrogen/Research Genetics, Carlsbad, CA). Pathways 2.0 (Research Genetics, Carlsbad, CA) and Bioconductor packages (http://www.bioconductor.org) were used to quantify the background intensity, normalize data (loess normalization), and determine actually expressed genes.
Keywords = Homo sapiens
Keywords = human micro vascular endothelial cell
Keywords = endostatin
Keywords = angiogenesis


title: HMVEC treated with PEDF

status: Public on Dec 31 2005

type: RNA

city: Padova

first: Silvio

last: Bicciato

address: via U. Bassi 59/b

department: Molecular Medicine

country: Italy

institute: University of Padova

description:
Total RNA was first isolated from HMVEC’s that had been incubated with Pigment Epithelium Derived Factor at IC50 concentration (PEDF, 0.4 nM) for 4 hr. Total RNA was isolated using TRIZOL solution (Invitrogen, Carlsbad, CA). Briefly, after treatment, cells were washed twice in PBS and incubated with TRIZOL for 5 minutes at room temperature. RNA was extracted with 200 ul chloroform per ml TRIZOL and precipitated with 100% isopropanol at -20ºC. The RNA pellet was resuspended in DEPC-treated water and stored at –80ºC until further use. Human cDNA microarrays (Research Genetics/Invitrogen, Carlsbad, CA) consisted of nylon membranes containing 4,132 named genes EST’s (GF211). The array also contained 192 spots of total genomic DNA that served as reference points for the image analysis software Pathways 2.0 (Research Genetics/Invitrogen, Carlsbad, CA) for normalization purposes and to verify the homogeneity of the hybridization. cDNA probes were made from 5 micro-g total RNA with α[33P]-dATP (Amersham Biosciences, Piscataway, NJ) by oligo dT-primed polymerization using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). Probes were purified by gel chromatography (BioSpin 6; BioRad Laboratories, Hercules, CA), boiled for about 3 min and allowed to cool to room temperature. The microarrays were pre-hybridized for no less than 2 hr and then hybridized overnight at 42 oC. Filters were washed with 2xSSC and 1 % SDS for 15 min at 50 oC, and then with 0.5xSSC and 1% SDS twice for a total of 30 min at room temperature. Washed filters were then exposed to phosphor imager screens, which were scanned by a Molecular Dynamics Storm Imager (Packard Instruments, Meriden, CT) at 600 dpi resolution. Scanned files containing the microarray were analyzed with Pathways software (Invitrogen/Research Genetics, Carlsbad, CA). Pathways 2.0 (Research Genetics, Carlsbad, CA) and Bioconductor packages (http://www.bioconductor.org) were used to quantify the background intensity, normalize data (loess normalization), and determine actually expressed genes.
Keywords = Homo sapiens
Keywords = human micro vascular endothelial cell
Keywords = Pigment Epithelium Derived Factor
Keywords = angiogenesis

molecule: total RNA

organism: Homo sapiens

source_name: Human micro vascular endothelial cells treated with PEDF


title: HMVEC Control of PEDF

description:
Control RNA was extracted from untreated HMVEC. Total RNA was isolated using TRIZOL solution (Invitrogen, Carlsbad, CA). Briefly, after treatment, cells were washed twice in PBS and incubated with TRIZOL for 5 minutes at room temperature. RNA was extracted with 200 ul chloroform per ml TRIZOL and precipitated with 100% isopropanol at -20ºC. The RNA pellet was resuspended in DEPC-treated water and stored at –80ºC until further use. Human cDNA microarrays (Research Genetics/Invitrogen, Carlsbad, CA) consisted of nylon membranes containing 4,132 named genes EST’s (GF211). The array also contained 192 spots of total genomic DNA that served as reference points for the image analysis software Pathways 2.0 (Research Genetics/Invitrogen, Carlsbad, CA) for normalization purposes and to verify the homogeneity of the hybridization. cDNA probes were made from 5 micro-g total RNA with α[33P]-dATP (Amersham Biosciences, Piscataway, NJ) by oligo dT-primed polymerization using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). Probes were purified by gel chromatography (BioSpin 6; BioRad Laboratories, Hercules, CA), boiled for about 3 min and allowed to cool to room temperature. The microarrays were pre-hybridized for no less than 2 hr and then hybridized overnight at 42 oC. Filters were washed with 2xSSC and 1 % SDS for 15 min at 50 oC, and then with 0.5xSSC and 1% SDS twice for a total of 30 min at room temperature. Washed filters were then exposed to phosphor imager screens, which were scanned by a Molecular Dynamics Storm Imager (Packard Instruments, Meriden, CT) at 600 dpi resolution. Scanned files containing the microarray were analyzed with Pathways software (Invitrogen/Research Genetics, Carlsbad, CA). Pathways 2.0 (Research Genetics, Carlsbad, CA) and Bioconductor packages (http://www.bioconductor.org) were used to quantify the background intensity, normalize data (loess normalization), and determine actually expressed genes.
Keywords = Homo sapiens
Keywords = human micro vascular endothelial cell
Keywords = Pigment Epithelium Derived Factor
Keywords = angiogenesis

source_name: Untreated human micro vascular endothelial cells


title: HMVEC Control of TNP-470

description:
Control RNA was extracted from untreated HMVEC. Total RNA was isolated using TRIZOL solution (Invitrogen, Carlsbad, CA). Briefly, after treatment, cells were washed twice in PBS and incubated with TRIZOL for 5 minutes at room temperature. RNA was extracted with 200 ul chloroform per ml TRIZOL and precipitated with 100% isopropanol at -20ºC. The RNA pellet was resuspended in DEPC-treated water and stored at –80ºC until further use. Human cDNA microarrays (Research Genetics/Invitrogen, Carlsbad, CA) consisted of nylon membranes containing 4,132 named genes EST’s (GF211). The array also contained 192 spots of total genomic DNA that served as reference points for the image analysis software Pathways 2.0 (Research Genetics/Invitrogen, Carlsbad, CA) for normalization purposes and to verify the homogeneity of the hybridization. cDNA probes were made from 5 micro-g total RNA with α[33P]-dATP (Amersham Biosciences, Piscataway, NJ) by oligo dT-primed polymerization using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). Probes were purified by gel chromatography (BioSpin 6; BioRad Laboratories, Hercules, CA), boiled for about 3 min and allowed to cool to room temperature. The microarrays were pre-hybridized for no less than 2 hr and then hybridized overnight at 42 oC. Filters were washed with 2xSSC and 1 % SDS for 15 min at 50 oC, and then with 0.5xSSC and 1% SDS twice for a total of 30 min at room temperature. Washed filters were then exposed to phosphor imager screens, which were scanned by a Molecular Dynamics Storm Imager (Packard Instruments, Meriden, CT) at 600 dpi resolution. Scanned files containing the microarray were analyzed with Pathways software (Invitrogen/Research Genetics, Carlsbad, CA). Pathways 2.0 (Research Genetics, Carlsbad, CA) and Bioconductor packages (http://www.bioconductor.org) were used to quantify the background intensity, normalize data (loess normalization), and determine actually expressed genes.
Keywords = Homo sapiens
Keywords = human micro vascular endothelial cell
Keywords = TNP-470
Keywords = angiogenesis


title: Germinal Center B cells; GC_50

status: Public on Aug 07 2008

city: RALEIGH

first: Dereje

last: Jima

state: NC

address: 850 Main Campus Drive

department: CHHE

country: USA

institute: NCSU

description: Gene expression data from Germinal Center B cells

hyb_protocol: Affymetrix protocol

scan_protocol: Affymetrix protocol

label: biotin

source_name: Germinal Center B cells

label_protocol: Affymetrix protocol

extract_protocol: CD19 selected B cells were sorted using flow cytometry

tag: Acutly resected tonsils from young patients undergoing routine tonsillectomy


title: HMVEC Control of RA

description:
Control RNA was extracted from untreated HMVEC. Total RNA was isolated using TRIZOL solution (Invitrogen, Carlsbad, CA). Briefly, after treatment, cells were washed twice in PBS and incubated with TRIZOL for 5 minutes at room temperature. RNA was extracted with 200 ul chloroform per ml TRIZOL and precipitated with 100% isopropanol at -20ºC. The RNA pellet was resuspended in DEPC-treated water and stored at –80ºC until further use. Human cDNA microarrays (Research Genetics/Invitrogen, Carlsbad, CA) consisted of nylon membranes containing 4,132 named genes EST’s (GF211). The array also contained 192 spots of total genomic DNA that served as reference points for the image analysis software Pathways 2.0 (Research Genetics/Invitrogen, Carlsbad, CA) for normalization purposes and to verify the homogeneity of the hybridization. cDNA probes were made from 5 micro-g total RNA with α[33P]-dATP (Amersham Biosciences, Piscataway, NJ) by oligo dT-primed polymerization using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). Probes were purified by gel chromatography (BioSpin 6; BioRad Laboratories, Hercules, CA), boiled for about 3 min and allowed to cool to room temperature. The microarrays were pre-hybridized for no less than 2 hr and then hybridized overnight at 42 oC. Filters were washed with 2xSSC and 1 % SDS for 15 min at 50 oC, and then with 0.5xSSC and 1% SDS twice for a total of 30 min at room temperature. Washed filters were then exposed to phosphor imager screens, which were scanned by a Molecular Dynamics Storm Imager (Packard Instruments, Meriden, CT) at 600 dpi resolution. Scanned files containing the microarray were analyzed with Pathways software (Invitrogen/Research Genetics, Carlsbad, CA). Pathways 2.0 (Research Genetics, Carlsbad, CA) and Bioconductor packages (http://www.bioconductor.org) were used to quantify the background intensity, normalize data (loess normalization), and determine actually expressed genes.
Keywords = Homo sapiens
Keywords = human micro vascular endothelial cell
Keywords = retinoic acid
Keywords = angiogenesis


title: HMVEC treated with TNP-470

description:
Total RNA was first isolated from HMVEC’s that had been incubated with TNP-470 (National Cancer Institute) at IC50 concentration (TNP, 1.5 nM) for 4 hr. Total RNA was isolated using TRIZOL solution (Invitrogen, Carlsbad, CA). Briefly, after treatment, cells were washed twice in PBS and incubated with TRIZOL for 5 minutes at room temperature. RNA was extracted with 200 ul chloroform per ml TRIZOL and precipitated with 100% isopropanol at -20ºC. The RNA pellet was resuspended in DEPC-treated water and stored at –80ºC until further use. Human cDNA microarrays (Research Genetics/Invitrogen, Carlsbad, CA) consisted of nylon membranes containing 4,132 named genes EST’s (GF211). The array also contained 192 spots of total genomic DNA that served as reference points for the image analysis software Pathways 2.0 (Research Genetics/Invitrogen, Carlsbad, CA) for normalization purposes and to verify the homogeneity of the hybridization. cDNA probes were made from 5 micro-g total RNA with α[33P]-dATP (Amersham Biosciences, Piscataway, NJ) by oligo dT-primed polymerization using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). Probes were purified by gel chromatography (BioSpin 6; BioRad Laboratories, Hercules, CA), boiled for about 3 min and allowed to cool to room temperature. The microarrays were pre-hybridized for no less than 2 hr and then hybridized overnight at 42 oC. Filters were washed with 2xSSC and 1 % SDS for 15 min at 50 oC, and then with 0.5xSSC and 1% SDS twice for a total of 30 min at room temperature. Washed filters were then exposed to phosphor imager screens, which were scanned by a Molecular Dynamics Storm Imager (Packard Instruments, Meriden, CT) at 600 dpi resolution. Scanned files containing the microarray were analyzed with Pathways software (Invitrogen/Research Genetics, Carlsbad, CA). Pathways 2.0 (Research Genetics, Carlsbad, CA) and Bioconductor packages (http://www.bioconductor.org) were used to quantify the background intensity, normalize data (loess normalization), and determine actually expressed genes.
Keywords = Homo sapiens
Keywords = human micro vascular endothelial cell
Keywords = TNP-470
Keywords = angiogenesis

source_name: Human micro vascular endothelial cells treated with TNP_470


title: Plasma B Cells; PC_30

description: Gene expression data from Plasma B cells

source_name: Plasma B cells


title: Germinal Center B cells; GC_43


title: Patient #35

status: Public on Jun 13 2003

description:
Blast cells have been obtained from the bone marrow of patient #35 affected by B-lymphoblastic leukemia. The blast cells and mononuclear cells have been isolated using a Lymphoprep™ density gradient (1.077 g/ml, Nycomed Pharma, Oslo, Norway) and cryo-preserved in liquid nitrogen (FCS 90% and 10% dimethylsulfoxide DMSO). Total RNA samples (8 ug each) were isolated using Trizol (Invitrogen). The samples were labeled using CyScribe First Strand cDNA Labeling Kit (Amersham Biosciences) as described by the manufacturer's instructions. Total RNA control samples (diagnosis) were labeled using Cy3 dUTP (Amersham Biosciences), and total RNA experimental samples (relapses) were labeled using Cy5 dUTP (Amersham Biosciences) during reverse transcription. For reverse transcription 8 ug of total RNA was mixed with Anchored oligo-dT at 70°C for 5 minutes, then add the labeling components in the following order: 4 uL of 5X CyScript Buffer, 2 uL of 0.1 M DTT, 1 uL of dUTP nucleotide mix, 1 uL dUTP CyDye-labeled nucleotide and 1 uL CyScript reverse transcriptase. The reaction mixtures were incubated at 42°C for 1.5 hours. After the reverse transcription reactions, the RNA templates were degraded by addition of 2 uL of 2.5 M NaOH and incubation at 37°C for 15 minutes. The alkaline conditions were neutralized by addition of 10 uL of 2 M HEPES. The cDNA samples were cleaned using a Microcon centrifugal filter device (Millipore) as described by the manufacturer’s instructions. The cleaned samples were concentrated in a speed vacufuge (Savant) for 10 minutes without heat. Experimental cDNA samples were then mixed with a control samples, dissolved in 240 uL salt based hybridization buffer (MWG-Biotech Laboratories), heated for 3 minutes at 95°C, and hybridized to Human 30K array A oligonucleotide microarrays (MWG-Biotech Laboratories). Hybridization occurred overnight at 42°C using wet hybridization chambers (MWG-Biotech Laboratories). The following day the arrays were washed for 5 minutes at room temperature in 2X SSC 0.1% SDS, then in 1X SSC and finally in 0.1X SSC. The arrays were dried by centrifugation and scanned using a ScanArray 4000 scanner (Packard Bioscence Instruments). The resulting data is downloaded and formatted in Excel (Microsoft), and analyzed using Matlab (The MathWorks, Inc.).
Keywords = Homo sapiens, B-lymphoblastic leukemia, diagnosis, relapse

source_name: Patient #35 blast cells at diagnosis

source_name: Patient #35 blast cells at relapse


title: Patient #53

description:
Blast cells have been obtained from the bone marrow of patient #53 affected by B-lymphoblastic leukemia. The blast cells and mononuclear cells have been isolated using a Lymphoprep™ density gradient (1.077 g/ml, Nycomed Pharma, Oslo, Norway) and cryo-preserved in liquid nitrogen (FCS 90% and 10% dimethylsulfoxide DMSO). Total RNA samples (8 ug each) were isolated using Trizol (Invitrogen). The samples were labeled using CyScribe First Strand cDNA Labeling Kit (Amersham Biosciences) as described by the manufacturer's instructions. Total RNA control samples (diagnosis) were labeled using Cy3 dUTP (Amersham Biosciences), and total RNA experimental samples (relapses) were labeled using Cy5 dUTP (Amersham Biosciences) during reverse transcription. For reverse transcription 8 ug of total RNA was mixed with Anchored oligo-dT at 70°C for 5 minutes, then add the labeling components in the following order: 4 uL of 5X CyScript Buffer, 2 uL of 0.1 M DTT, 1 uL of dUTP nucleotide mix, 1 uL dUTP CyDye-labeled nucleotide and 1 uL CyScript reverse transcriptase. The reaction mixtures were incubated at 42°C for 1.5 hours. After the reverse transcription reactions, the RNA templates were degraded by addition of 2 uL of 2.5 M NaOH and incubation at 37°C for 15 minutes. The alkaline conditions were neutralized by addition of 10 uL of 2 M HEPES. The cDNA samples were cleaned using a Microcon centrifugal filter device (Millipore) as described by the manufacturer’s instructions. The cleaned samples were concentrated in a speed vacufuge (Savant) for 10 minutes without heat. Experimental cDNA samples were then mixed with a control samples, dissolved in 240 uL salt based hybridization buffer (MWG-Biotech Laboratories), heated for 3 minutes at 95°C, and hybridized to Human 30K array A oligonucleotide microarrays (MWG-Biotech Laboratories). Hybridization occurred overnight at 42°C using wet hybridization chambers (MWG-Biotech Laboratories). The following day the arrays were washed for 5 minutes at room temperature in 2X SSC 0.1% SDS, then in 1X SSC and finally in 0.1X SSC. The arrays were dried by centrifugation and scanned using a ScanArray 4000 scanner (Packard Bioscence Instruments). The resulting data is downloaded and formatted in Excel (Microsoft), and analyzed using Matlab (The MathWorks, Inc.).
Keywords = Homo sapiens, B-lymphoblastic leukemia, diagnosis, relapse

source_name: Patient #53 blast cells at diagnosis

source_name: Patient #53 blast cells at relapse


title: Patient #54

description:
Blast cells have been obtained from the bone marrow of patient #54 affected by B-lymphoblastic leukemia. The blast cells and mononuclear cells have been isolated using a Lymphoprep™ density gradient (1.077 g/ml, Nycomed Pharma, Oslo, Norway) and cryo-preserved in liquid nitrogen (FCS 90% and 10% dimethylsulfoxide DMSO). Total RNA samples (8 ug each) were isolated using Trizol (Invitrogen). The samples were labeled using CyScribe First Strand cDNA Labeling Kit (Amersham Biosciences) as described by the manufacturer's instructions. Total RNA control samples (diagnosis) were labeled using Cy3 dUTP (Amersham Biosciences), and total RNA experimental samples (relapses) were labeled using Cy5 dUTP (Amersham Biosciences) during reverse transcription. For reverse transcription 8 ug of total RNA was mixed with Anchored oligo-dT at 70°C for 5 minutes, then add the labeling components in the following order: 4 uL of 5X CyScript Buffer, 2 uL of 0.1 M DTT, 1 uL of dUTP nucleotide mix, 1 uL dUTP CyDye-labeled nucleotide and 1 uL CyScript reverse transcriptase. The reaction mixtures were incubated at 42°C for 1.5 hours. After the reverse transcription reactions, the RNA templates were degraded by addition of 2 uL of 2.5 M NaOH and incubation at 37°C for 15 minutes. The alkaline conditions were neutralized by addition of 10 uL of 2 M HEPES. The cDNA samples were cleaned using a Microcon centrifugal filter device (Millipore) as described by the manufacturer’s instructions. The cleaned samples were concentrated in a speed vacufuge (Savant) for 10 minutes without heat. Experimental cDNA samples were then mixed with a control samples, dissolved in 240 uL salt based hybridization buffer (MWG-Biotech Laboratories), heated for 3 minutes at 95°C, and hybridized to Human 30K array A oligonucleotide microarrays (MWG-Biotech Laboratories). Hybridization occurred overnight at 42°C using wet hybridization chambers (MWG-Biotech Laboratories). The following day the arrays were washed for 5 minutes at room temperature in 2X SSC 0.1% SDS, then in 1X SSC and finally in 0.1X SSC. The arrays were dried by centrifugation and scanned using a ScanArray 4000 scanner (Packard Bioscence Instruments). The resulting data is downloaded and formatted in Excel (Microsoft), and analyzed using Matlab (The MathWorks, Inc.).
Keywords = Homo sapiens, B-lymphoblastic leukemia, diagnosis, relapse

source_name: Patient #54 blast cells at diagnosis

source_name: Patient #54 blast cells at relapse


title: Patient #31

description:
Blast cells have been obtained from the bone marrow of patient #31 affected by B-lymphoblastic leukemia.
The blast cells and mononuclear cells have been isolated using a Lymphoprep™ density gradient (1.077 g/ml, Nycomed Pharma, Oslo, Norway) and cryo-preserved in liquid nitrogen (FCS 90% and 10% dimethylsulfoxide DMSO).
Total RNA samples (8 ug each) were isolated using Trizol (Invitrogen). The samples were labeled using CyScribe First Strand cDNA Labeling Kit (Amersham Biosciences) as described by the manufacturer's instructions. Total RNA control samples (diagnosis) were labeled using Cy3 dUTP (Amersham Biosciences), and total RNA experimental samples (relapses) were labeled using Cy5 dUTP (Amersham Biosciences) during reverse transcription. For reverse transcription 8 ug of total RNA was mixed with Anchored oligo-dT at 70°C for 5 minutes, then add the labeling components in the following order: 4 uL of 5X CyScript Buffer, 2 uL of 0.1 M DTT, 1 uL of dUTP nucleotide mix, 1 uL dUTP CyDye-labeled nucleotide and 1 uL CyScript reverse transcriptase. The reaction mixtures were incubated at 42°C for 1.5 hours. After the reverse transcription reactions, the RNA templates were degraded by addition of 2 uL of 2.5 M NaOH and incubation at 37°C for 15 minutes. The alkaline conditions were neutralized by addition of 10 uL of 2 M HEPES. The cDNA samples were cleaned using a Microcon centrifugal filter device (Millipore) as described by the manufacturer’s instructions. The cleaned samples were concentrated in a speed vacufuge (Savant) for 10 minutes without heat. Experimental cDNA samples were then mixed with a control samples, dissolved in 240 uL salt based hybridization buffer (MWG-Biotech Laboratories), heated for 3 minutes at 95°C, and hybridized to Human 30K array A oligonucleotide microarrays (MWG-Biotech Laboratories). Hybridization occurred overnight at 42°C using wet hybridization chambers (MWG-Biotech Laboratories). The following day the arrays were washed for 5 minutes at room temperature in 2X SSC 0.1% SDS, then in 1X SSC and finally in 0.1X SSC. The arrays were dried by centrifugation and scanned using a ScanArray 4000 scanner (Packard Bioscence Instruments). The resulting data have been preprocessed using QuantArray (Packard Bioscence Instruments), downloaded and formatted in Excel (Microsoft), and analyzed using Matlab (The MathWorks, Inc.).
Keywords = Homo sapiens, B-lymphoblastic leukemia, diagnosis, relapse

source_name: Patient #31 blast cells at diagnosis

source_name: Patient #31 blast cells at relapse


title: Patient #56

description:
Blast cells have been obtained from the bone marrow of patient #56 affected by B-lymphoblastic leukemia. The blast cells and mononuclear cells have been isolated using a Lymphoprep™ density gradient (1.077 g/ml, Nycomed Pharma, Oslo, Norway) and cryo-preserved in liquid nitrogen (FCS 90% and 10% dimethylsulfoxide DMSO). Total RNA samples (8 ug each) were isolated using Trizol (Invitrogen). The samples were labeled using CyScribe First Strand cDNA Labeling Kit (Amersham Biosciences) as described by the manufacturer's instructions. Total RNA control samples (diagnosis) were labeled using Cy3 dUTP (Amersham Biosciences), and total RNA experimental samples (relapses) were labeled using Cy5 dUTP (Amersham Biosciences) during reverse transcription. For reverse transcription 8 ug of total RNA was mixed with Anchored oligo-dT at 70°C for 5 minutes, then add the labeling components in the following order: 4 uL of 5X CyScript Buffer, 2 uL of 0.1 M DTT, 1 uL of dUTP nucleotide mix, 1 uL dUTP CyDye-labeled nucleotide and 1 uL CyScript reverse transcriptase. The reaction mixtures were incubated at 42°C for 1.5 hours. After the reverse transcription reactions, the RNA templates were degraded by addition of 2 uL of 2.5 M NaOH and incubation at 37°C for 15 minutes. The alkaline conditions were neutralized by addition of 10 uL of 2 M HEPES. The cDNA samples were cleaned using a Microcon centrifugal filter device (Millipore) as described by the manufacturer’s instructions. The cleaned samples were concentrated in a speed vacufuge (Savant) for 10 minutes without heat. Experimental cDNA samples were then mixed with a control samples, dissolved in 240 uL salt based hybridization buffer (MWG-Biotech Laboratories), heated for 3 minutes at 95°C, and hybridized to Human 30K array A oligonucleotide microarrays (MWG-Biotech Laboratories). Hybridization occurred overnight at 42°C using wet hybridization chambers (MWG-Biotech Laboratories). The following day the arrays were washed for 5 minutes at room temperature in 2X SSC 0.1% SDS, then in 1X SSC and finally in 0.1X SSC. The arrays were dried by centrifugation and scanned using a ScanArray 4000 scanner (Packard Bioscence Instruments). The resulting data is downloaded and formatted in Excel (Microsoft), and analyzed using Matlab (The MathWorks, Inc.).
Keywords = Homo sapiens, B-lymphoblastic leukemia, diagnosis, relapse

source_name: Patient #56 blast cells at diagnosis

source_name: Patient #56 blast cells at relapse


title: Patient #65

description:
Blast cells have been obtained from the bone marrow of patient #65 affected by B-lymphoblastic leukemia.
The blast cells and mononuclear cells have been isolated using a Lymphoprep™ density gradient (1.077 g/ml, Nycomed Pharma, Oslo, Norway) and cryo-preserved in liquid nitrogen (FCS 90% and 10% dimethylsulfoxide DMSO).
Total RNA samples (8 ug each) were isolated using Trizol (Invitrogen). The samples were labeled using CyScribe First Strand cDNA Labeling Kit (Amersham Biosciences) as described by the manufacturer's instructions. Total RNA control samples (diagnosis) were labeled using Cy3 dUTP (Amersham Biosciences), and total RNA experimental samples (relapses) were labeled using Cy5 dUTP (Amersham Biosciences) during reverse transcription. For reverse transcription 8 ug of total RNA was mixed with Anchored oligo-dT at 70°C for 5 minutes, then add the labeling components in the following order: 4 uL of 5X CyScript Buffer, 2 uL of 0.1 M DTT, 1 uL of dUTP nucleotide mix, 1 uL dUTP CyDye-labeled nucleotide and 1 uL CyScript reverse transcriptase. The reaction mixtures were incubated at 42°C for 1.5 hours. After the reverse transcription reactions, the RNA templates were degraded by addition of 2 uL of 2.5 M NaOH and incubation at 37°C for 15 minutes. The alkaline conditions were neutralized by addition of 10 uL of 2 M HEPES. The cDNA samples were cleaned using a Microcon centrifugal filter device (Millipore) as described by the manufacturer’s instructions. The cleaned samples were concentrated in a speed vacufuge (Savant) for 10 minutes without heat. Experimental cDNA samples were then mixed with a control samples, dissolved in 240 uL salt based hybridization buffer (MWG-Biotech Laboratories), heated for 3 minutes at 95°C, and hybridized to Human 30K array A oligonucleotide microarrays (MWG-Biotech Laboratories). Hybridization occurred overnight at 42°C using wet hybridization chambers (MWG-Biotech Laboratories). The following day the arrays were washed for 5 minutes at room temperature in 2X SSC 0.1% SDS, then in 1X SSC and finally in 0.1X SSC. The arrays were dried by centrifugation and scanned using a ScanArray 4000 scanner (Packard Bioscence Instruments). The resulting data have been preprocessed using QuantArray (Packard Bioscence Instruments), downloaded and formatted in Excel (Microsoft), and analyzed using Matlab (The MathWorks, Inc.).
Keywords = Homo sapiens, B-lymphoblastic leukemia, diagnosis, relapse

source_name: Patient #65 blast cells at diagnosis

source_name: Patient #65 blast cells at relapse


title: Patient #32

description:
Blast cells have been obtained from the bone marrow of patient #32 affected by B-lymphoblastic leukemia. The blast cells and mononuclear cells have been isolated using a Lymphoprep™ density gradient (1.077 g/ml, Nycomed Pharma, Oslo, Norway) and cryo-preserved in liquid nitrogen (FCS 90% and 10% dimethylsulfoxide DMSO). Total RNA samples (8 ug each) were isolated using Trizol (Invitrogen). The samples were labeled using CyScribe First Strand cDNA Labeling Kit (Amersham Biosciences) as described by the manufacturer's instructions. Total RNA control samples (diagnosis) were labeled using Cy3 dUTP (Amersham Biosciences), and total RNA experimental samples (relapses) were labeled using Cy5 dUTP (Amersham Biosciences) during reverse transcription. For reverse transcription 8 ug of total RNA was mixed with Anchored oligo-dT at 70°C for 5 minutes, then add the labeling components in the following order: 4 uL of 5X CyScript Buffer, 2 uL of 0.1 M DTT, 1 uL of dUTP nucleotide mix, 1 uL dUTP CyDye-labeled nucleotide and 1 uL CyScript reverse transcriptase. The reaction mixtures were incubated at 42°C for 1.5 hours. After the reverse transcription reactions, the RNA templates were degraded by addition of 2 uL of 2.5 M NaOH and incubation at 37°C for 15 minutes. The alkaline conditions were neutralized by addition of 10 uL of 2 M HEPES. The cDNA samples were cleaned using a Microcon centrifugal filter device (Millipore) as described by the manufacturer’s instructions. The cleaned samples were concentrated in a speed vacufuge (Savant) for 10 minutes without heat. Experimental cDNA samples were then mixed with a control samples, dissolved in 240 uL salt based hybridization buffer (MWG-Biotech Laboratories), heated for 3 minutes at 95°C, and hybridized to Human 30K array A oligonucleotide microarrays (MWG-Biotech Laboratories). Hybridization occurred overnight at 42°C using wet hybridization chambers (MWG-Biotech Laboratories). The following day the arrays were washed for 5 minutes at room temperature in 2X SSC 0.1% SDS, then in 1X SSC and finally in 0.1X SSC. The arrays were dried by centrifugation and scanned using a ScanArray 4000 scanner (Packard Bioscence Instruments). The resulting data is downloaded and formatted in Excel (Microsoft), and analyzed using Matlab (The MathWorks, Inc.).
Keywords = Homo sapiens, B-lymphoblastic leukemia, diagnosis, relapse

source_name: Patient #32 blast cells at diagnosis

source_name: Patient #32 blast cells at relapse


title: Patient #52

description: Blast cells have been obtained from the bone marrow of patient #52 affected by B-lymphoblastic leukemia. The blast cells and mononuclear cells have been isolated using a Lymphoprep™ density gradient (1.077 g/ml, Nycomed Pharma, Oslo, Norway) and cryo-preserved in liquid nitrogen (FCS 90% and 10% dimethylsulfoxide DMSO). Total RNA samples (8 ug each) were isolated using Trizol (Invitrogen). The samples were labeled using CyScribe First Strand cDNA Labeling Kit (Amersham Biosciences) as described by the manufacturer's instructions. Total RNA control samples (diagnosis) were labeled using Cy3 dUTP (Amersham Biosciences), and total RNA experimental samples (relapses) were labeled using Cy5 dUTP (Amersham Biosciences) during reverse transcription. For reverse transcription 8 ug of total RNA was mixed with Anchored oligo-dT at 70°C for 5 minutes, then add the labeling components in the following order: 4 uL of 5X CyScript Buffer, 2 uL of 0.1 M DTT, 1 uL of dUTP nucleotide mix, 1 uL dUTP CyDye-labeled nucleotide and 1 uL CyScript reverse transcriptase. The reaction mixtures were incubated at 42°C for 1.5 hours. After the reverse transcription reactions, the RNA templates were degraded by addition of 2 uL of 2.5 M NaOH and incubation at 37°C for 15 minutes. The alkaline conditions were neutralized by addition of 10 uL of 2 M HEPES. The cDNA samples were cleaned using a Microcon centrifugal filter device (Millipore) as described by the manufacturer’s instructions. The cleaned samples were concentrated in a speed vacufuge (Savant) for 10 minutes without heat. Experimental cDNA samples were then mixed with a control samples, dissolved in 240 uL salt based hybridization buffer (MWG-Biotech Laboratories), heated for 3 minutes at 95°C, and hybridized to Human 30K array A oligonucleotide microarrays (MWG-Biotech Laboratories). Hybridization occurred overnight at 42°C using wet hybridization chambers (MWG-Biotech Laboratories). The following day the arrays were washed for 5 minutes at room temperature in 2X SSC 0.1% SDS, then in 1X SSC and finally in 0.1X SSC. The arrays were dried by centrifugation and scanned using a ScanArray 4000 scanner (Packard Bioscence Instruments). The resulting data is downloaded and formatted in Excel (Microsoft), and analyzed using Matlab (The MathWorks, Inc.).

source_name: Patient #52 blast cells at diagnosis

source_name: Patient #52 blast cells at relapse