title: LPS+R848 2h 1

description:
This sample is also known as IOSI05-007 and IRBG3
Sample_characteristics: Monocyte derived dendritic cells were generated by culturing human monocytes isolated from buffy coats using CD14 microbeads (Miltenyi)
Sample_data_processing: Affymetrix GCOS 1.2
Sample_extract_protocol: cells were harvested and lysed using Trizol (Invitrogen). An additional RNA clean-up step using the RNAeasy purification kit (Qiagen).
Sample_growth_protocol_ch: cultured for five days in 10cm tissue culture dishes in RPMI with 10% fetal bovine serum (Hyclone) supplemented with GM-CSF 50ng/ml and IL-4 10 ng/ml. On day 5 MoDCs were harvested and aliquoted into 6 well plate at 0.5 106 cells/ml, 1.5 106 cells/well.
Sample_hyb_protocol: standard Affymetrix procedures, washes performed using Fluidics Station 400
Sample_label: biotin
Sample_label_protocol: Affymetrix GeneChip Expression 3’ Amplification One-Cycle Target Labeling kit from about 5ug of total RNA
Sample_molecule: total RNA
Sample_scan_protocol: standard Affymetrix procedures using a GeneChip Scanner 3000
Sample_treatment_protocol_ch: after 2h of incubation at 37°C cells were stimulated with 10ng/ml E. coli  0111:B4 LPS Ultra-Pure (Invivogen) and 2.5?g/ml R848 (GLSynthesis) for 2h


title: Donor 5 1g 4 hr

status: Public on Aug 25 2005

type: RNA

city: Bethesda

first: Jim

last: Boonyaratanakornkit

state: MD

address: 50 South Dr, Bldg 50, Rm 6513

department: LID

country: USA

institute: NIH - NIAID

description:
Peripheral blood leukocytes of three human donors were isolated from blood bank buffy-coat preparations by Ficoll-Hypaque density gradient centrifugation, and T-cells were purified with high affinity negative selection human CD3+ T-cell enrichment columns.  The cells were resuspended in RPMI-1640 with 10% FCS with approximately 3-8 millions cells/mL at room temperature overnight.  T-cells were then either unactivated (1g 0 hr), activated (1g 4 hr) with a final concentration of 5 mg/mL Con A + 4 mg/mL anti-CD28 antibody and incubated for 4 hours at 37°C, or loaded onto a RPM rotating at 60°/s.  T-cells loaded on the RPM were cultured for 2 hours prior to collection or activation to allow equilibration of cells to the new environment.  After incubation, 1 mL of 4 M guanidinium isothiocyanate in 750 mM sodium citrate buffer, pH 7, with N-lauroylsarcosine and ß-mercaptoethanol was added to lyse cells and stabilize RNA.  Lysates were frozen at -80°C.
Lot batch = 2001996

molecule: total RNA

organism: Homo sapiens

source_name: T-cells, peripheral blood


title: Donor 4 vg 4 hr


title: Donor 4 1g 4 hr


title: Donor 5 1g 0 hr


title: R848 2h 1

status: Public on Sep 01 2005

city: Bellinzona

first: Francesco

last: Bertoni

state: Ticino

address: Via Vela 6

department: Experimental Oncology

country: Switzerland

institute: Institute of Southern Switzerland

description:
This sample is also known as IOSI05-006 and IRBG2
Sample_characteristics: Monocyte derived dendritic cells were generated by culturing human monocytes isolated from buffy coats using CD14 microbeads (Miltenyi)
Sample_data_processing: Affymetrix GCOS 1.2
Sample_extract_protocol: cells were harvested and lysed using Trizol (Invitrogen). An additional RNA clean-up step using the RNAeasy purification kit (Qiagen).
Sample_growth_protocol_ch: cultured for five days in 10cm tissue culture dishes in RPMI with 10% fetal bovine serum (Hyclone) supplemented with GM-CSF 50ng/ml and IL-4 10 ng/ml. On day 5 MoDCs were harvested and aliquoted into 6 well plate at 0.5 106 cells/ml, 1.5 106 cells/well.
Sample_hyb_protocol: standard Affymetrix procedures, washes performed using Fluidics Station 400
Sample_label: biotin
Sample_label_protocol: Affymetrix GeneChip Expression 3’ Amplification One-Cycle Target Labeling kit from about 5ug of total RNA
Sample_molecule: total RNA
Sample_scan_protocol: standard Affymetrix procedures using a GeneChip Scanner 3000
Sample_treatment_protocol_ch: after 2h of incubation at 37°C cells were stimulated  with 2.5?g/ml R848 (GLSynthesis) for 2h

source_name: Monocyte derived dendritic cells


title: Donor 2 vg 0 hr


title: Donor 4 vg 0 hr


title: Donor 4 1g 0 hr


title: untreated 3

description:
This sample is also known as IOSI05-025 and IRBDL97
Sample_characteristics: Monocyte derived dendritic cells were generated by culturing human monocytes isolated from buffy coats using CD14 microbeads (Miltenyi)
Sample_data_processing: Affymetrix GCOS 1.2
Sample_extract_protocol: cells were harvested and lysed using Trizol (Invitrogen). An additional RNA clean-up step using the RNAeasy purification kit (Qiagen).
Sample_growth_protocol_ch: cultured for five days in 10cm tissue culture dishes in RPMI with 10% fetal bovine serum (Hyclone) supplemented with GM-CSF 50ng/ml and IL-4 10 ng/ml. On day 5 MoDCs were harvested and aliquoted into 6 well plate at 0.5 106 cells/ml, 1.5 106 cells/well.
Sample_hyb_protocol: standard Affymetrix procedures, washes performed using Fluidics Station 400
Sample_label: biotin
Sample_label_protocol: Affymetrix GeneChip Expression 3’ Amplification One-Cycle Target Labeling kit from about 5ug of total RNA
Sample_molecule: total RNA
Sample_scan_protocol: standard Affymetrix procedures using a GeneChip Scanner 3000
Sample_treatment_protocol_ch: after 2h of incubation at 37°C cells were harvested and RNA extracted


title: Donor 2 vg 4 hr


title: ScN2a_6

status: Public on Aug 24 2005

city: Neuherberg

first: Martin

last: Irmler

state: Bayern

address: Ingolstaedter Landstrasse 1

department: Institute of Experimental Genetics

country: Germany

institute: Helmholtz Zentrum München GmbH

description:
Mouse neuroblastoma (N2a) cells are available from the American Tissue Culture Collection (ATCC, number CCL-131). To generate cells expressing 3F4-tagged PrP, cells were stably transfected with Mo3F4 PrP in a pcDNA3.1/Zeo vector (Invitrogen, Leek, The Netherlands) and stable transfectants selected using Zeozine 300 ug/ml medium (Gilch et al, 2001, EMBO 20: 3957-3966).
RNA was isolated from control and infected cell lines by the Trizol method.
20µg total RNA was used for reverse transcription and indirectly labelled with Cy3 or Cy5 fluorescent dye according the TIGR protocol. Labelled cDNA was dissolved in 30µl hybridisation buffer (6x SSC, 0,5% SDS 5 fold Denhardt's solution and 50% formamide) and mixed with 30µl of reference cDNA solution labelled with the second dye. This hybridisation mixture was placed on a pre hybriddised microarray, covered with cover slip, placed into a hybridisation chamber (Genomatix) and immersed in a thermostatic bath at 42°C for at least 16 hours.
Slides were scanned for both Cy3 and Cy5 with a GenePix 4000A and images were processed by GenePix Pro 3.0. Maen intensities with substracted background were normalised so that the median of their rations has became 1

Keywords = prion
Keywords = neuroblastoma
Keywords = N2a

organism: Mus musculus


title: Donor 2 1g 4 hr

description:
Peripheral blood leukocytes of three human donors were isolated from blood bank buffy-coat preparations by Ficoll-Hypaque density gradient centrifugation, and T-cells were purified with high affinity negative selection human CD3+ T-cell enrichment columns.  The cells were resuspended in RPMI-1640 with 10% FCS with approximately 3-8 millions cells/mL at room temperature overnight.  T-cells were then either unactivated (1g 0 hr), activated (1g 4 hr) with a final concentration of 5 mg/mL Con A + 4 mg/mL anti-CD28 antibody and incubated for 4 hours at 37°C, or loaded onto a RPM rotating at 60°/s.  T-cells loaded on the RPM were cultured for 2 hours prior to collection or activation to allow equilibration of cells to the new environment.  After incubation, 1 mL of 4 M guanidinium isothiocyanate in 750 mM sodium citrate buffer, pH 7, with N-lauroylsarcosine and ß-mercaptoethanol was added to lyse cells and stabilize RNA.  Lysates were frozen at -80°C.
Lot batch = 2001663


title: ScGT1_3

description:
A detailed description of this cell lines and the infection with prions, can be found in Schätzl et al., 1997. By single cell cloning consecutive subclones of ScGT1 cells were prepared.
RNA was isolated from control and infected cell lines by the Trizol method.
20µg total RNA was used for reverse transcription and indirectly labelled with Cy3 or Cy5 fluorescent dye according the TIGR protocol. Labelled cDNA was dissolved in 30µl hybridisation buffer (6x SSC, 0,5% SDS 5 fold Denhardt's solution and 50% formamide) and mixed with 30µl of reference cDNA solution labelled with the second dye. This hybridisation mixture was placed on a pre hybriddised microarray, covered with cover slip, placed into a hybridisation chamber (Genomatix) and immersed in a thermostatic bath at 42°C for at least 16 hours.
Slides were scanned for both Cy3 and Cy5 with a GenePix 4000A and images were processed by GenePix Pro 3.0. Maen intensities with substracted background were normalised so that the median of their rations has became 1

Keywords = prion
Keywords = hypothalamus
Keywords = GT1


title: ScGT1_2


title: ScN2a_2


title: ScGT1_1


title: ScN2a_4


title: Donor 2 1g 0 hr

description:
Peripheral blood leukocytes of three human donors were isolated from blood bank buffy-coat preparations by Ficoll-Hypaque density gradient centrifugation, and T-cells were purified with high affinity negative selection human CD3+ T-cell enrichment columns.  The cells were resuspended in RPMI-1640 with 10% FCS with approximately 3-8 million cells/mL at room temperature overnight.  T-cells were then either unactivated (1g 0 hr), activated (1g 4 hr) with a final concentration of 5 µg/mL Con A + 4 µg/mL anti-CD28 antibody and incubated for 4 hours at 37°C, or loaded onto a RPM rotating at 60°/s.  T-cells loaded on the RPM were cultured for 2 hours prior to collection or activation to allow equilibration of cells to the new environment.  After incubation, 1 mL of 4 M guanidinium isothiocyanate in 750 mM sodium citrate buffer, pH 7, with N-lauroylsarcosine and ß-mercaptoethanol was added to lyse cells and stabilize RNA.  Lysates were frozen at –80°C.
Lot batch = 2001663


title: ScN2a_5


title: ScN2a_7

description:
Mouse neuroblastoma (N2a) cells are available from the American Tissue Culture Collection (ATCC, number CCL-131). To generate cells expressing 3F4-tagged PrP, cells were stably transfected with Mo3F4 PrP in a pcDNA3.1/Zeo vector (Invitrogen, Leek, The Netherlands) and stable transfectants selected using Zeozine 300 ug/ml medium (Gilch et al, 2001, EMBO 20: 3957-3966).
RNA was isolated from control and infected cell lines by the Trizol method.
20µg total RNA was used for reverse transcription and indirectly labelled with Cy3 or Cy5 fluorescent dye according the TIGR protocol. Labelled cDNA was dissolved in 30µl hybridisation buffer (6x SSC, 0,5% SDS 5 fold Denhardt's solution and 50% formamide) and mixed with 30µl of reference cDNA solution labelled with the second dye. This hybridisation mixture was placed on a pre hybriddised microarray, covered with cover slip, placed into a hybridisation chamber (Genomatix) and immersed in a thermostatic bath at 42°C for at least 16 hours.
Slides were scanned for both Cy3 and Cy5 with a GenePix 4000A and images were processed by GenePix Pro 3.0. Maen intensities with substracted background were normalised so that the median of their rations has became 1
Keywords = prion
Keywords = neuroblastoma
Keywords = N2a


title: ScGT1_4

description:
A detailed description of this cell lines and the infection with prions, can be found in Schätzl et al., 1997. By single cell cloning consecutive subclones of ScGT1 cells were prepared.
RNA was isolated from control and infected cell lines by the Trizol method.
20µg total RNA was used for reverse transcription and indirectly labelled with Cy3 or Cy5 fluorescent dye according the TIGR protocol. Labelled cDNA was dissolved in 30µl hybridisation buffer (6x SSC, 0,5% SDS 5 fold Denhardt's solution and 50% formamide) and mixed with 30µl of reference cDNA solution labelled with the second dye. This hybridisation mixture was placed on a pre hybriddised microarray, covered with cover slip, placed into a hybridisation chamber (Genomatix) and immersed in a thermostatic bath at 42°C for at least 16 hours.
Slides were scanned for both Cy3 and Cy5 with a GenePix 4000A and images were processed by GenePix Pro 3.0. Maen intensities with substracted background were normalised so that the median of their rations has became 1
Keywords = prion
Keywords = hypothalamus
Keywords = GT1


title: ScN2a_3


title: static (control) experiment array

status: Public on Aug 22 2005

city: Dublin

first: Ruaidhri

middle: Patrick

last: Kirwan

state: Leinster

address: Belfield

department: Conway Institute of Biomolecular and Biomedical Research & School of Medicine and Medical Science

country: Ireland

institute: University College Dublin

description: control array data for comparison with an experiment exposing LC cells to 24 hours of 15% 1 Hz cyclical mechanical strain.

data_processing: Robust multichip average (RMA)

label: biotin

source_name: GFAP-negative lamina cribrosa cells

tag: GFAP-negative

tag: normal human donors

tag: optic nerve head tissue

tag: lamina cribrosa layer

tag: primary cultures


title: CBMNC.LPS+.2

status: Public on Aug 20 2005

city: Heidelberg

first: Lutz

last: Koch

address: Im Neuenheimer Feld 153

department: Department of Paediatrics

institute: University of Heidelberg

description: to evaluate in vitro activation of the neonatal and adult immune system, gene expression patterns were compared in mononuclear cells from cord and adult peripheral blood without and after incubation with 5 µg/ml Lipopolysaccharide (LPS).

hyb_protocol: Biotinylated cRNA was heated for 5 min at 95°C, allowing RNA to breakdown to small fragments according to Affymetrix protocols. Fragmented cRNA (20 µg) was subjected to oligonucleotide hybridization (Hybridization Oven; Affymetrix) to the human Genome Focus Genechip (Affymetrix).

data_processing: software package R, version 1.9.1, libraries gcrma and limma of the Bioconductor Project

label: Biotin

source_name: neonatal cord blood mononunclear cells after LPS-treatment

label_protocol: Biotinylated labeling cRNA was conducted by in vitro transcription using the BioArray High Yield RNA Transcript Labeling System (ENZO). cRNA was purified by GeneChip Sample Cleanup Module (Affymetrix).

extract_protocol: Total RNA was extracted from 1.5 x 107 CBMNC or APBMNC with RNeasy Kit (Qiagen). Quantity and purity of extracted RNA was checked by RNA LabChip® Kit (Agilent).

tag: Peripheral blood was obtained by venipuncture from healthy adult volunteers. Blood samples from umbilical cords of the placentas of healthy, full-term infants after scheduled caesarean section or vaginal delivery were obtained by puncture of the cord immediately after delivery


title: CBMNC.LPS-.1

source_name: neonatal cord blood mononunclear cells without LPS-treatment


title: APBMNC.LPS+.3

source_name: adult peripheral blood mononuclear cells after LPS-treatment


title: CBMNC.LPS-.2


title: CBMNC.LPS-.3


title: APBMNC.LPS+.2


title: CBMNC.LPS+.1


title: CBMNC.LPS+.3


title: APBMNC.LPS-.1

data_processing: software package R, version 1.9.1,  libraries gcrma and limma of the Bioconductor Project

source_name: adult peripheral blood mononuclear cells without LPS-treatment


title: APBMNC.LPS+.1


title: APBMNC.LPS-.3


title: APBMNC.LPS-.2


title: 9 Fairplay day 2 chip 3

status: Public on Aug 19 2005

city: Odense

first: Mads

last: Thomassen

address: Soender Boulevard 9

department: Clinical Genetics (KKA)

country: Denmark

institute: Odense University Hospital

description: Self-hybridisation

hyb_protocol: Hybridization and washing were performed identically for all arrays. Each cDNA or cRNA mixture was adjusted with 4x SSC, 0.1 % SDS, and 0.1µg/µl sonicated salmon sperm DNA ( ) to a final volume of 80 µl. Samples were denaturated for 2 min at 95 °C, then spun at 14,000 rpm for 5 min. Samples were hybridized to microarray slides using lifter slips (Erie) as covers and placed in CMT-Hybridization chambers (Corning Inc., Corning, NY). To maintain humidity inside the chambers, 10 µl of DEPC water was added to the two reservoir wells. After tightly sealing of the chambers, hybridization was done overnight at 42 °C in a water bath for 17 hours. After hybridization, slides were removed from the chambers and lifter slips were gently removed from the slides by soaking in 4x SSC in a wash station (TeleChem International Inc., Sunnyvale, USA). Slides were washed by immersion into 2x SSC, 0.1 SDS twice at hybridization temperature for 5 min, in 0.2x SSC and 0.1x SSC each of them at room temperature for 1 min, and dried by centrifugation at 1000 rpm for 5 min

scan_protocol: Detection of Cy3 and Cy5 fluorescence signals were performed on an ArrayWoRxe CCD scanner (Applied Precision) and images were generated for the Cy3 and Cy5 channels with the accompanying Array WoRxe software.

data_processing: VSN, R Biocondutor

source_name: Reference RNA

tag: Reference RNA


title: 9 Fairplay day 3 chip 1

description: Self-hybridisation


title: 9 Fairplay day 2 chip 2

description: Self-hybridisation


title: 9 Fairplay day 1 chip 3

description: Self-hybridisation


title: 9 messageAmp day 3 chip 2

description: Self-hybridisation


title: 9 Fairplay day 3 chip 2

description: Self-hybridisation


title: 9 Fairplay day 1 chip 2

description: Self-hybridisation


title: 9 Fairplay day 3 chip 3

description: Self-hybridisation


title: 9 Fairplay day 2 chip 1

description: Self-hybridisation


title: 9 messageAmp day 3 chip 3

description: Self-hybridisation


title: 9 Fairplay day 1 chip 1

description: Self-hybridisation


title: 9 messageAmp day 3 chip 1

description: Self-hybridisation


title: 9 messageAmp day 2 chip 3

description: Self-hybridisation


title: 9 messageAmp day 1 chip 1

description: Self-hybridisation


title: 9 messageAmp day 1 chip 2

description: Self-hybridisation


title: 5 Fairplay day 1 chip 3

description: Self-hybridisation

data_processing: VSN, R


title: 9 messageAmp day 2 chip 2

description: Self-hybridisation


title: 9 messageAmp day 1 chip 3

description: Self-hybridisation


title: 5 Fairplay day 1 chip 5

description: Self-hybridisation


title: 5 MessageAmp day 1 chip 4

description: Self-hybridisation


title: 5 MessageAmp day 1 chip 5

description: Self-hybridisation


title: 5 Fairplay day 1 chip 4

description: Self-hybridisation


title: 9 messageAmp day 2 chip 1

description: Self-hybridisation


title: 5 Fairplay day 1 chip 1

description: Self-hybridisation


title: 5 MessageAmp day 1 chip 1

description: Self-hybridisation

hyb_protocol: Hybridization and washing were performed identically for all arrays. Each cDNA or cRNA mixture was adjusted with 4x SSC, 0.1 % SDS, and 0.1µg/µl sonicated salmon sperm DNA ( ) to a final volume of 80 µl. Samples were denaturated for 2 min at 95 °C, then spun at 14,000 rpm for 5 min.  Samples were hybridized to microarray slides using lifter slips (Erie) as covers and placed in CMT-Hybridization chambers (Corning Inc., Corning, NY). To maintain humidity inside the chambers, 10 µl of DEPC water was added to the two reservoir wells. After tightly sealing of the chambers, hybridization was done overnight at 42 °C in a water bath for 17 hours. After hybridization, slides were removed from the chambers and lifter slips were gently removed from the slides by soaking in 4x SSC in a wash station (TeleChem International Inc., Sunnyvale, USA). Slides were washed by immersion into 2x SSC, 0.1 SDS twice at hybridization temperature for 5 min, in 0.2x SSC and 0.1x SSC each of them at room temperature for 1 min, and dried by centrifugation at 1000 rpm for 5 min


title: 5 MessageAmp day 1 chip 2

description: Self-hybridisation


title: 5 MessageAmp day 1 chip 3

description: Self-hybridisation


title: 5 Fairplay day 1 chip 2

description: Self-hybridisation


title: 10MTHF2B

status: Public on Aug 13 2005

city: Wageningen

first: Jaap

last: Keijer

address: Bornsesteeg 45

country: Netherlands

institute: RIKILT Institute of Food Safety

description: The 3DNA Array 350 HS Expression array detection kit for microarrays (Cy3/Cy5 Kit, Genisphere, Hatfield, USA) was used for both cDNA synthesis and microarray hybridization. Briefly, 5 µg of total RNA from each sample (4 x 3) was reverse transcribed in duplo using 5 pmol capture sequence primer. The Cy5 specific primer was used for the individual samples and the Cy3 specific primer was used for the reference samples, consisting of a pool of RNA extracted from all samples. In total, 4 x 3 x 2 = 24 reactions were performed for both Cy5 and Cy3 samples. For all individual reactions, total RNA (5 µg in 1-10 µl) and RT primer (1 µl) were brought to a final volume of 11 µl with nuclease free water and incubated at 80 degrees C. Afterwards, 1 µl of Superase-In was added and a reaction mix consisting of 4 µl 5x SuperScript II first strand buffer, 1 µl dNTP mix (10 mM each for dATP, dCTP, dGTp, dTTP), 2 µl 0.1 M DTT and 1 µl Superscript II enzyme (200 units) (Invitrogen). The reaction was incubated at 42 degrees C for 2 hours. The reaction was terminated by adding 3.5 µl 0.5 M NaOH/50 mM EDTA and incubation at 65 degrees C for 10 minutes. The reaction was neutralized with 5 µl 1 M Tris-HCl, pH 7.5. The Cy5 and Cy3 reactions were then combined per sample, purified with the Qiaquick PCR purification kit (Westburg) and precipitated overnight at -20 degrees C using 1/10 volume of 3 M sodium-acetate (pH 5.2) and 2.5 volumes of 96% ethanol.

data_processing: ArrayVision for visualisation of the array, GeneMathsXT for Rikilt normalisation and further data-analysis

source_name: human H29 cell line reference

tag: total mRNA of all the experiments

source_name: human H29 cell line experiment

tag: experiment treated with 10ng/ml MTHF (natural folic acid), second cell culture, second of the duplo


title: 10PGA1B

tag: experiment treated with 10ng/ml PGA (synthetic folic acid), first cell culture, second of the duplo


title: 10MTHF1B

tag: experiment treated with 10ng/ml MTHF (natural folic acid), first cell culture, second of the duplo


title: 10PGA3B

tag: experiment treated with 10ng/ml PGA (synthetic folic acid), third cell culture, second of the duplo


title: ScN2a_1


title: 10MTHF1A

tag: experiment treated with 10ng/ml MTHF (natural folic acid), first cell culture, first of the duplo


title: 10MTHF3B

tag: experiment treated with 10ng/ml MTHF (natural folic acid), third cell culture, second of the duplo


title: 10PGA3A

tag: experiment treated with 10ng/ml PGA (synthetic folic acid), third cell culture, first of the duplo


title: 10PGA1A

tag: experiment treated with 10ng/ml PGA (synthetic folic acid), first cell culture, first of the duplo


title: 10MTHF2A

tag: experiment treated with 10ng/ml MTHF (natural folic acid), second cell culture, first of the duplo


title: 100MTHF1A

tag: experiment treated with 100ng/ml MTHF (natural folic acid), first cell culture, first of the duplo


title: 100MTHF2A

tag: experiment treated with 100ng/ml MTHF (natural folic acid), second cell culture, first of the duplo


title: 100PGA1A

tag: experiment treated with 100ng/ml PGA (synthetic folic acid), first cell culture, first of the duplo


title: 100MTHF3A

tag: experiment treated with 100ng/ml MTHF (natural folic acid), third cell culture, first of the duplo


title: 100MTHF2B

tag: experiment treated with 100ng/ml MTHF (natural folic acid), second cell culture, second of the duplo


title: 100MTHF1B

tag: experiment treated with 100ng/ml MTHF (natural folic acid), first cell culture, second of the duplo


title: 100MTHF3B

tag: experiment treated with 100ng/ml MTHF (natural folic acid), third cell culture, second of the duplo


title: GD11:12_conotruncus_FolR1(wt)_c

status: Public on Feb 20 2006

city: Houston

first: Huiping

last: Zhu

state: TX

address: 2121 W. Holcombe Blvd

department: Center for Environmental and Genetic Medicine

institute: Institute of Biosciences and Technology, TAMUSHSC

description: The median of the expression values for each sample at the specified timepoint was used to generate the effective expression ratios representing the impact of FolR1 gene ablation on embryonic development in mice during the critical phase of aorticopulmonary septation.

data_processing: R 2.0.0, scaled to 1.0

label: Fluor 647-streptavidin

tag: LmBc_FolR1 wild type, gestational day

value: GD11:12, tissue:conotruncus


title: Colitis 10, UC10

status: Public on Aug 11 2005

city: Kiel

first: Robert

last: Haesler

address: Schittenhelmstr. 12

department: IKMB / 1st Dept. of Medicine

institute: University Clinic of Schleswig Holstein

description:
Pathohistological and clinical examination of Patient UC10 (43 year-old male) yielded to diagnosis of ulcerative colitis.
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).

source_name: sigmoid colon biopsy


title: Colitis 04, UC04

description:
Pathohistological and clinical examination of Patient UC04 (31 year-old male) yielded to diagnosis of ulcerative colitis
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: Colitis 03, UC03

description:
Pathohistological and clinical examination of Patient UC03 (27 year-old male)yielded to diagnosis of ulcerative colitis
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: Colitis 09, UC09

description:
Pathohistological and clinical examination of Patient UC09 (29 year-old female) yielded to diagnosis of ulcerative colitis.
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: 100PGA2A

tag: total mRNA of all the experiments

tag: experiment treated with 100ng/ml PGA (synthetic folic acid), second cell culture, first of the duplo


title: 100PGA3B

description: The 3DNA  Array 350 HS Expression array detection kit for microarrays (Cy3/Cy5 Kit, Genisphere, Hatfield, USA) was used for both cDNA synthesis and microarray hybridization. Briefly, 5 µg of total RNA from each sample (4 x 3) was reverse transcribed in duplo using 5 pmol capture sequence primer. The Cy5 specific primer was used for the individual samples and the Cy3 specific primer was used for the reference samples, consisting of a pool of RNA extracted from all samples. In total, 4 x 3 x 2 = 24 reactions were performed for both Cy5 and Cy3 samples. For all individual reactions, total RNA (5 µg in 1-10 µl) and RT primer (1 µl) were brought to a final volume of 11 µl with nuclease free water and incubated at 80 degrees C. Afterwards, 1 µl of Superase-In was added and a reaction mix consisting of 4 µl 5x SuperScript II first strand buffer, 1 µl dNTP mix (10 mM each for dATP, dCTP, dGTp, dTTP), 2 µl 0.1 M DTT and 1 µl Superscript II enzyme (200 units) (Invitrogen). The reaction was incubated at 42 degrees C for 2 hours. The reaction was terminated by adding 3.5 µl 0.5 M NaOH/50 mM EDTA and incubation at 65 degrees C for 10 minutes. The reaction was neutralized with 5 µl 1 M Tris-HCl, pH 7.5. The Cy5 and Cy3 reactions were then combined per sample, purified with the Qiaquick PCR purification kit (Westburg) and precipitated overnight at -20 degrees C using 1/10 volume of 3 M sodium-acetate (pH 5.2) and 2.5 volumes of 96% ethanol.

source_name: Human H29 cell line ref

source_name: Human H29 cell line exp

tag: experiment treated with 100ng/ml PGA, third cell culture. second sample of the duplo


title: Colitis 02, UC02

description:
Pathohistological and clinical examination of Patient UC02 (38 year-old male) yielded to diagnosis of ulcerative colitis
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: 100PGA2B

tag: experiment treated with 100ng/ml PGA (synthetic folic acid), second cell culture, second of the duplo


title: ORF clr3-myc binding map(K080D#12)

type: genomic

city: Stockholm

first: Karl

last: Ekwall

address: Alfred Nobels Alle 7

country: Sweden

institute: Karolinska Inst

description: For HDAC binding (Clr3-myc and Sir2- myc), ChIP was performed according to Kurdistani et al (2002), also with the modification to the protocol of leaving out the cold PBS wash. We used 1.5–3.0 mg 9E10 monoclonal antibody against Myc(Sigma) for each IP.

molecule: genomic DNA

organism: Schizosaccharomyces pombe

source_name: CHIP IP clr3-myc

tag: clr3-myc sample

source_name: CHIP Input

tag: CHIP input clr3-myc


title: Colitis 05, UC05

description:
Pathohistological and clinical examination of Patient UC05 (29 year-old male) yielded to diagnosis of ulcerative colitis.
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: Colitis 08, UC08

description:
Pathohistological and clinical examination of Patient UC08 (51 year-old male) yielded to diagnosis of ulcerative colitis.
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: 100PGA1B

tag: experiment treated with 100ng/ml PGA (synthetic folic acid), first cell culture, second of the duplo


title: Colitis 06, UC06

description:
Pathohistological and clinical examination of Patient UC06 (38 year-old female) yielded to diagnosis of ulcerative colitis.
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: Colitis 01, UC01

description:
Pathohistological and clinical examination of Patient UC01 (29 year-old male) yielded to diagnosis of ulcerative colitis
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: Crohn 09, CD09

description:
Pathohistological and clinical examination of Patient CD09 (50 year-old male) yielded to diagnosis of Crohn's disease.
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: Crohn 10, CD10

description:
Pathohistological and clinical examination of Patient CD10 (18 year-old female) yielded to diagnosis of Crohn's disease.
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: IGR clr3-myc binding map(K080D#12)


title: ORF clr3-myc binding map(K080D#11)


title: IGR clr3-myc binding map(K080D#11)


title: Crohn 04, CD04

description:
Pathohistological and clinical examination of Patient CD04 (32 year-old female) yielded to diagnosis of Crohn's disease.
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible.
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: Crohn 03, CD03

description:
Pathohistological and clinical examination of Patient CD03 (19 year-old male) yielded to diagnosis of Crohn's disease.
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible.
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: Crohn 02, CD02

description:
Pathohistological and clinical examination of Patient CD02 (38 year-old female) yielded to diagnosis of Crohn's disease.
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible.
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: Crohn 01, CD01

description:
Pathohistological and clinical examination of Patient CD01 (20 year-old male) yielded to diagnosis of Crohn's disease.
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible.
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: ORF sir2-myc binding map(#4)

source_name: CHIP IP sir2-myc

tag: sir2-myc sample

tag: CHIP input sir2-myc


title: Colitis 07, UC07

description:
Pathohistological and clinical examination of Patient UC07 (33 year-old male) yielded to diagnosis of ulcerative colitis.
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: ORF clr3-myc binding map(#12)


title: ORF clr3-myc binding map(#5)


title: IGR sir2-myc binding map(#4)


title: Control 01, NC01

description:
Pathohistological and clinical examination of Patient NC01 (44 year-old male) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).


title: IGR clr3-myc binding map(#5)


title: ORF sir2-myc binding map(#14)


title: IGR sir2-myc binding map(#14)


title: ORF sir2-myc binding map(B160E #4)


title: ORF sir2-myc binding map(B160E #13)


title: IGR clr3-myc binding map(#12)


title: 100PGA3A

description: The 3DNA Array 350 HS Expression array detection kit for microarrays (Cy3/Cy5 Kit, Genisphere, Hatfield, USA) was used for both cDNA synthesis and microarray hybridization. Briefly, 5 µg of total RNA from each sample (4 x 3) was reverse transcribed in duplo using 5 pmol capture sequence primer. The Cy5 specific primer was used for the individual samples and the Cy3 specific primer was used for the reference samples, consisting of a pool of RNA extracted from all samples. In total, 4 x 3 x 2 = 24 reactions were performed for both Cy5 and Cy3 samples. For all individual reactions, total RNA (5 µg in 1-10 µl) and RT primer (1 µl) were brought to a final volume of 11 µl with nuclease free water and incubated at 80 degrees C. Afterwards, 1 µl of Superase-In was added and a reaction mix consisting of 4 µl 5x SuperScript II first strand buffer, 1 µl dNTP mix (10 mM each for dATP, dCTP, dGTp, dTTP), 2 µl 0.1 M DTT and 1 µl Superscript II enzyme (200 units) (Invitrogen). The reaction was incubated at 42 degrees C for 2 hours. The reaction was terminated by adding 3.5 µl 0.5 M NaOH/50 mM EDTA and incubation at 65 degrees C for 10 minutes. The reaction was neutralized with 5 µl 1 M Tris-HCl, pH 7.5. The Cy5 and Cy3 reactions were then combined per sample, purified with the Qiaquick PCR purification kit (Westburg) and precipitated overnight at -20 degrees C using 1/10 volume of 3 M sodium-acetate (pH 5.2) and 2.5 volumes of 96% ethanol.

tag: experiment treated with 100ng/ml PGA (synthetic folic acid), third cell culture, first of the duplo


title: IGR clr3-myc binding map(#14)


title: IGR sir2-myc binding map(B160E #13)


title: Control 09, NC09

description:
Pathohistological and clinical examination of Patient NC09 (76 year-old female) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.
Target Labeling and Hybridization procedure:
100-250 ng mRNA was isolated from total RNA for each patient using the Oligotex kit (Qiagen) according to the manufacturer’s instructions.  Prior to hybridization, 20ng of Arabidopsis thaliana DNA template was labeled with a-33P dCTP (10 microCi/microL; Amersham) during a random priming labeling reaction (pd(N)6, Amersham) (Feinberg and Vogelstein, 1983).  Reversed transcribed cDNA from patient targets were prepared in a final volume of 30microL as described in detail previously, with the exception that the labeling reaction was carried out at 42°C for 50 min (Eickhoff et al. 2000).  RNA was hydrolyzed under alkaline conditions by the addition of 3.5 microL of 3M NaOH and incubation for 20 min at 68°C. The samples were neutralized with the addition of 10 microL of 1M Tris-HCl (pH 7.4) and 10.5 microL of 1M HCl.  Unincorporated radioactive nucleotides were removed from both control and patient targets by filtration through Microspin G-50 columns according to the product manual (Amersham).
Prior to each hybridization reaction, unused filters were initially washed in 1 X SSC; 0.1% SDS at room temperature for 10 min (X 2) followed by three washes in 0.1 X SSC; 0.1% SDS for 20 min at 80°C.  These washes eliminated the potential problem for unbound or loosely bound probe cDNA from interfering with the hybridization reaction.  Each cDNA filter was pre-wetted with deionized water and placed in a hybridization bottle (3.5 X 25 cm).  The water was completely poured off and the filter was pre-hybridized for 2 hrs at 50°C in a 10 mL of hybridization solution containing 7% SDS, 50% formamide, 5 X SCC, 2% blocking reagent (Roche Diagnostics), 50mM sodium phosphate (pH 7.0) and denatured herring sperm DNA (50microg/microL).  The labeled target was added to the filters in 5 mL of the same preheated solution and hybridization of the filters was performed overnight (24 hrs) at 42°C.  Following overnight hybridization, membranes were removed from the bottles and washed in 1 X SSC/0.1% SDS buffer for 10 min at room temperature followed by two washes for 30 min in 0.2 X SSC/0.1% SDS at 65°C.  Excess moisture was removed by briefly placing the membranes on filter paper (Whatman) and the damp filters were wrapped in commercial grade plastic wrap.  Filters were exposed ~24 hrs to imaging plates (BAS-MS 2325, Fujifilm, Japan) and scanning was carried out at a resolution of 50microns on a FLA-3000G imaging system (Fujifilm, Japan). After hybridization, filters were individually stripped in a boiling solution of 0.5% SDS for 5 min, taken off the heat and agitated for an additional 3 hrs at room temperature and stored damp wrapped in plastic wrap at –20ºC for 7-8 weeks (half-life of 33P is 25.4 days). Prior to re-use, filters were re-scanned as above to ensure that residual radioactivity was negligible
Spot quantitation:
An exposure time of ~24 h was sufficient to yield signals from cDNA clones, yet keeping the data overflow to a mimimum.  Images were quantified using VisualGrid® software V3.4 (http://www.gpc-biotech.com). Each grid was manually adjusted to the image such that the linear correlation coefficient between duplicate spots was greater than 0.984. ID (Location) 95A01 - 96P24 contained spotting buffer only and were flaged as empty / set to zero.
References:
Eickhoff, H. et al. Tissue gene expression analysis using arrayed normalized cDNA libraries. Genome Res 10, 1230-40. (2000).
Feinberg, A.P. & Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13. (1983).