title: GH Treated Male-Male(cy3-cy5) #3-RL

description:
The genetic expression profile for one male and one female continuous GH treated male liver sample is given as a lowess normalized ratio (f=0.2).  This result is from a single random pairing of one of four males and one of four GH treated males.  The purpose of this pairwise comparison is to highlight the GH-dependent differences in rat liver gene expression.

Technical Aspects:  Adult male and female Fischer 344 rats (8-10 weeks of age) were untreated or were treated with rat growth hormone (GH) (NIDDK, rGH-B-14-SIAFP) given as a continuous infusion using an Alzet osmotic minipump delivering 2 ug GH/100g body weight/h for 7 days.  Intact male and female rats were killed and their livers were collected, frozen in liquid N2 and stored at –80ºC.  Total liver RNA was isolated from frozen liver samples using Trizol reagent (Invitrogen).  Poly(A) RNA was then isolated using  a commercial kit (Ambion).  RNA samples having an A260/A280 ratio >= 1.8 were deemed suitable for use in subsequent analyses.  Total liver RNA and poly(A) RNA samples were treated with DNase I (Ambion).  Poly(A) RNA purified from individual livers was labeled with Cy3-dUTP or Cy5-dUTP (PerkinElmer) in a reverse transcription reaction using 1 ug poly(A) RNA and Superscript II (RNase H mutant) enzyme (Invitrogen) at 39oC for 2 hr.  Unincorporated dNTPs were removed using the Qiaquick PCR-purification kit (Qiagen).  Cy5-labeled male liver cDNA was mixed with Cy3-labeled female or Cy3-labeled GH-treated male liver cDNA followed by co-hybridization of the mixed cDNA population to the microarray for 18 hr at 42oC.  Arrays were washed according to the manufacturer’s protocol.  RNA from each of four male, female, and GH-treated male livers was converted to cDNA and labeled in duplicate.  One series of eight hybridizations (four male + female liver cDNA pairings, and four male + male with GH cDNA pairings)  was carried out on MWG Rat Liver arrays.  A second series containing the same eight hybridizations was carried out on MWG Rat 5K arrays.  The fluorescent cDNA on each microarray was detected using a GenePix 4000B array scanner (Axon Instruments).  The data was preprocessed using GenePix Pro 3.0 (Axon Instruments) and analyzed in GeneSpring 6.0 (Silicon Genetics).  Any spot where there was no fluorescence is reported as 0.  The normalized average ratio is given for all duplicates.

Keywords = Growth hormone
Keywords = liver sexual dimorphism
Keywords = cytochrome P450
Keywords = liver gene expression
Keywords = dual channel cDNA microarray


title: Gene expression changes induced by Acyclic Retinoid (GPL511)

status: Public on Apr 03 2004

type: RNA

city: Tokyo

first: Motoyuki

last: Otsuka

department: Department of Gastroenterology

country: Japan

institute: University of Tokyo

description: Total RNA was isolated following the Isogen procedure (Nippon-gene, Tokyo, Japan) from HepG2 cells with and without ACR treatment for 48 hours. Fluorescent cDNA probes (Cy5 and Cy3-labelled) were prepared from a 30 micro g total RNA isolated from HepG2 with ACR treatment and from a 30 micro g control total RNA isolated from HepG2 cells without ACR treatment, respectively. The different fluorescent-labeled probes were mixed and applied to a microarray following incubation at 42 degrees overnight under humidified conditions. Fluorescent images of the hybridized microarrays were scanned with a fluorescence laser confocal slide scanner (Affymetrix 428 Array Scanner, Affymetrix, Santa Clara, CA).  The images were analyzed using ImaGene 4.2 software (BioDiscovery, Marina Del Rey, CA) according to the manufacturer’s instructions.

molecule: total RNA

organism: Homo sapiens

source_name: 48 hours ACR treated HepG2

source_name: control (non-treated) HepG2


title: LNCaP 1 HR Time Point

status: Public on Jun 24 2004

city: Gaithersburg

first: Jeff

last: Kiefer

state: MD

address: 20 Firstfield Rd,  Suite 110

department: CDDL

country: USA

institute: Translational Genomics Research Institute (TGen)

description: LNCaP prostate cancer cells were treated with and without 100ug/ml Emetine Dihydrochloride Hydrate (Fluka, Buchs, Swizerland). Both Emetine-treated and untreated control cells were then incubated at 37°C for 10 hours. After the incubation, Actinomycin D (Sigma-Aldrich, St Louis, MO) with the final concentration of 5ug/ml, was added to the treated and untreated cells to stop new transcription. The 1 hour time point was harvested and cell pellets were snap-frozen and mRNA extracted by using FastTrack kit (Invitrogen) according to the manufacturer’s instructions. The untreated sample was hybridized against the Emetine-treated equivalent. Four ug of untreated mRNA was labeled with Cy5-dUTP and four ug of Emetine-treated mRNA with Cy3-dUTP (Amersham Biosciences, Piscataway, NJ) as previously described (Mousses et al, 2000). Average intensities of the tumor samples were divided by the average intensities of the reference sample at each microarray spot after background intensity subtraction. Within-slide normalization was performed with ratio statistics method using housekeeping genes as described previously (Chen et al, 1997). The data were quality filtered with ratio quality method (Chen et al, 2002), which computes a quality value for each ratio. The scale for the quality values is from zero (poor quality) to one (good quality). Here, all ratios having quality value below 0.5 were discarded from the subsequent analysis.

source_name: LNCaP 1 HR Time Point Emetine Treated

source_name: LNCaP 1 HR Time Point Un-Treated


title: Occipital #2

status: Public on Mar 27 2004

city: Kodaira

first: Nobuo

last: Funatsu

state: Tokyo

address: 4-1-1 Ogawahigashi

department: Cellular Biology

institute: National Institute of Neuroscience, NCNP

description: Dissection of E16.5 cortex for RNA extraction.   Cortex Hemispheres of E16.5 mice were divided into the medial (R1), dorsal (R2), ventral (R3), lateral (R4), and occipital regions (R5).   Preparation of RNA and Synthesis of cDNA. Poly A+ RNA was obtained through twice column procedures with oligo dT cellulose column (New England Biolabs, Beverly, MA) and was used for synthesis of cDNA. 1ug of polyA+ RNA was converted into double-stranded cDNA by reverse transcription using ReverTra Ace (ToYoBo, Japan) with oligo dT(24) primers containing the T7 RNA polymerase promotor sequence (Amersham Pharmacia Biotech, Japan). The integrity of RNA was confirmed by agarose electrophoresis and RNA contents were analyzed by measuring the optical density at 260 nm.  Target preparation and array processing. The following procedures including preparation of cRNA targets, hybridization, washing, staining, and scanning were performed as described in the Expression Analysis Technical Manual (Affymetrix, CA). Briefly, biotin-labeled antisense cRNA targets were synthesized in in vitro transcription reaction system (IVT) using cDNA as template (ENZO BioArrayTM HighYieldTM RNA Transcription Labeling kit with T7 RNA polymerase, Enzo, NY). The cRNA targets were size-fragmented to 35-200 bp in a buffer containing potassium and magnesium acetate at 94°C for 35 minutes and were used for hybridization as cRNA targets. The hybridization cocktail [containing 10 ug of target cRNA, control oligo BS (Affymetrix), and Eukaryotic Hybridization controls (Affymetrix)] was hybridized with a mouse U74A array at 42°C for 16 hours in GeneChip Hybridization Oven 640. Washing and staining were performed after hybridization under the fluidics station protocol, EuGE-WS2, on GeneChip Fluidics Station 400. Then, we scanned the array twice on HP GeneArrayTM scanner. Experiments were repeated twice with independent dissection.  Data analysis. To quantify the expression level of a transcript, the average difference value (Adv) was calculated from the intensities against the probe cells using the global methods of normalization by Microarray Suite ver. 4.0 (Affymetrix).

organism: Mus musculus

source_name: E16.5 mouse cortex


title: Rostral #2


title: Ventral #2

description:
Dissection of E16.5 cortex for RNA extraction.   Cortex Hemispheres of E16.5 mice were divided into the medial (R1), dorsal (R2), ventral (R3), lateral (R4), and occipital regions (R5).   Preparation of RNA and Synthesis of cDNA. Poly A+ RNA was obtained through twice column procedures with oligo dT cellulose column (New England Biolabs, Beverly, MA) and was used for synthesis of cDNA. 1ug of polyA+ RNA was converted into double-stranded cDNA by reverse transcription using ReverTra Ace (ToYoBo, Japan) with oligo dT(24) primers containing the T7 RNA polymerase promotor sequence (Amersham Pharmacia Biotech, Japan). The integrity of RNA was confirmed by agarose electrophoresis and RNA contents were analyzed by measuring the optical density at 260 nm.  Target preparation and array processing. The following procedures including preparation of cRNA targets, hybridization, washing, staining, and scanning were performed as described in the Expression Analysis Technical Manual (Affymetrix, CA). Briefly, biotin-labeled antisense cRNA targets were synthesized in in vitro transcription reaction system (IVT) using cDNA as template (ENZO BioArrayTM HighYieldTM RNA Transcription Labeling kit with T7 RNA polymerase, Enzo, NY). The cRNA targets were size-fragmented to 35-200 bp in a buffer containing potassium and magnesium acetate at 94 °C for 35 minutes and were used for hybridization as cRNA targets. The hybridization cocktail [containing 10 ug of target cRNA, control oligo BS (Affymetrix), and Eukaryotic Hybridization controls (Affymetrix)] was hybridized with a mouse U74A array at 42 °C for 16 hours in GeneChip Hybridization Oven 640. Washing and staining were performed after hybridization under the fluidics station protocol, EuGE-WS2, on GeneChip Fluidics Station 400. Then, we scanned the array twice on HP GeneArrayTM scanner. Experiments were repeated twice with independent dissection.  Data analysis. To quantify the expression level of a transcript, the average difference value (Adv) was calculated from the intensities against the probe cells using the global methods of normalization by Microarray Suite ver. 4.0 (Affymetrix).
Keywords = mouse
Keywords = cortex
Keywords = development
Keywords = regional
Keywords = gene expression
Keywords = medial
Keywords = dorsal
Keywords = lateral
Keywords = rostral
Keywords = occipital


title: Rostral #1


title: Ventral #1


title: Occipital #1


title: Dorsal #1


title: 4-NP 2

status: Public on Mar 20 2004

city: El Paso

first: William

last: Baldwin

state: TX

address: 500 W. University Ave.

department: Biological Sciences

institute: Unversity of Texas at El Paso

description:
Fifty micrograms of RNA from each mouse liver sample were enriched for poly A+ RNA using the AtlasTM Pure Total RNA Labeling system (Clontech, Palo Alto, CA), and reverse transcribed in the presence of [33P]dATP, dCTP, dGTP, dTTP and AMV reverse transcriptase to produce cDNA probes.  The probes were purified using the ProbeQuant™ G-50 Micro Columns (Amersham Pharmacia Biotech Inc., Piscataway, NJ), and then denatured in the presence of Cot-1 DNA.  The Clontech AtlasTM Mouse 1.2 Arrays (7853-1)(Palo Alto, CA) were prehybridized in 5 ml of ExpressHyb (Clontech) and 0.5 mg of heat denatured sheared salmon sperm for 30 minutes at 68C prior to probe incubation.  The probes were added and incubated overnight at 68C degrees before washing four times for 30 minutes each time with 2X SSC, 1% SDS, then once with 0.1X SSC, 0.5% SDS for 30 minutes, and once with 2X SSC for 5 minutes at 68oC.  Blots were visualized by placing them on a K-screen for 3-4 days (Bio-Rad, Hercules, CA).
Arrays were visualized from the K-screen, and analyzed and quantified with a phosphoimager (Molecular Imager FX, Bio-Rad, Hercules, CA) and the ResGen PathwaysTM Universal Microarray Analysis SoftwareTM (Research Genetics, Carlsbad, CA).  Local background was subtracted automatically from the individual spot intensities, and spot intensities were normalized to the mean intensity of all spots on the array.  This is called datapoint normalization on the ResGen Pathways Microarray Analysis SoftwareTM.  Normalized gene intensities on the arrays probed with 4-NP treated mouse livers and untreated mouse liver cDNA were compared, and fold differences were calculated.
Keywords = 4-nonylphenol

source_name: mouse liver


title: control 2


title: control 1


title: Dorsal #2


title: Medial #2


title: Medial #1


title: Female-Male(cy3-cy5) #1-RL

city: Boston

first: David

middle: J.

last: Waxman

state: MA

address: 5 Cummington Mall

department: Department of Biology and Bioinformatics Program

institute: Boston University

description:
The genetic expression profile for one male and one female rat liver sample is given as a lowess normalized ratio (f=0.2).  This result is from a single random pairing of one of four males and one of four females.  The purpose of this pairwise comparison is to highlight the sex-dependent differences in rat liver gene expression.

Technical Aspects:  Adult male and female Fischer 344 rats (8-10 weeks of age) were untreated or were treated with rat growth hormone (GH) (NIDDK, rGH-B-14-SIAFP) given as a continuous infusion using an Alzet osmotic minipump delivering 2 ug GH/100g body weight/h for 7 days.  Intact male and female rats were killed and their livers were collected, frozen in liquid N2 and stored at –80ºC.  Total liver RNA was isolated from frozen liver samples using Trizol reagent (Invitrogen).  Poly(A) RNA was then isolated using  a commercial kit (Ambion).  RNA samples having an A260/A280 ratio >= 1.8 were deemed suitable for use in subsequent analyses.  Total liver RNA and poly(A) RNA samples were treated with DNase I (Ambion).  Poly(A) RNA purified from individual livers was labeled with Cy3-dUTP or Cy5-dUTP (PerkinElmer) in a reverse transcription reaction using 1 ug poly(A) RNA and Superscript II (RNase H mutant) enzyme (Invitrogen) at 39oC for 2 hr.  Unincorporated dNTPs were removed using the Qiaquick PCR-purification kit (Qiagen).  Cy5-labeled male liver cDNA was mixed with Cy3-labeled female or Cy3-labeled GH-treated male liver cDNA followed by co-hybridization of the mixed cDNA population to the microarray for 18 hr at 42oC.  Arrays were washed according to the manufacturer’s protocol.  RNA from each of four male, female, and GH-treated male livers was converted to cDNA and labeled in duplicate.  One series of eight hybridizations (four male + female liver cDNA pairings, and four male + male with GH cDNA pairings)  was carried out on MWG Rat Liver arrays.  A second series containing the same eight hybridizations was carried out on MWG Rat 5K arrays.  The fluorescent cDNA on each microarray was detected using a GenePix 4000B array scanner (Axon Instruments).  The data was preprocessed using GenePix Pro 3.0 (Axon Instruments) and analyzed in GeneSpring 6.0 (Silicon Genetics).  Any spot where there was no fluorescence is reported as 0.  The normalized average ratio is given for all duplicates.

Keywords = Growth hormone
Keywords = liver sexual dimorphism
Keywords = cytochrome P450
Keywords = liver gene expression
Keywords = dual channel cDNA microarray

organism: Rattus norvegicus

source_name: frozen adult female rat liver tissue prepared using Trizol

source_name: frozen adult male rat liver tissue prepared using Trizol


title: 4-NP 1


title: anaerobic_wild_rep2

status: Public on Mar 10 2004

city: La Jolla

first: Jinseong

last: Cheong

state: CA

institute: University of California at San Diego

description: The purpose of this study is to investigate the changes of global gene expression in E. coli during an oxygen shift.  All cultures were grown under aerobic or anaerobic conditions in M9 minimal media supplemented with glucose.  Samples were RNA-stabilized using Qiagen RNAProtect Bacterial Reagent, and total RNA was isolated from exponentially growing cells using a Qiagen RNeasy mini kit (protocols available at www1.qiagen.com).  The RNA (10 µg) was then used as the template for cDNA synthesis, the product of which was fragmented, labelled, and hybridized to an Affymetrix E. coli Antisense Genome Array, which was washed and scanned to obtain an image.  All of these steps were performed according to Affymetrix protocols (available at www.affymetrix.com).

organism: Escherichia coli

source_name: anaerobic growth of wild-type strain on M9 media with glucose (reference)


title: anaerobic_wild_rep3


title: anaerobic_wild_rep4


title: anaerobic_soxS_rep3

source_name: anaerobic growth of soxS knock-out strain on M9 media with glucose


title: anaerobic_wild_rep1


title: anaerobic_oxyR_rep3

source_name: anaerobic growth of oxyR knock-out strain on M9 media with glucose


title: anaerobic_arcAfnr_rep3

source_name: anaerobic growth of arcA/fnr double knock-out strain on M9 media with glucose


title: anaerobic_soxS_rep2


title: anaerobic_fnr_rep1

source_name: anaerobic growth of fnr knock-out strain on M9 media with glucose


title: anaerobic_fnr_rep3


title: anaerobic_arcA_rep2

source_name: anaerobic growth of arcA knock-out strain on M9 media with glucose


title: anaerobic_fnr_rep2


title: anaerobic_arcA_rep3


title: anaerobic_oxyR_rep2


title: anaerobic_oxyR_rep1


title: anaerobic_arcA_rep1


title: anaerobic_soxS_rep1


title: anaerobic_appY_rep2

source_name: anaerobic growth of appY knock-out strain on M9 media with glucose


title: anaerobic_appY_rep3


title: anaerobic_arcAfnr_rep1


title: aerobic_wild_rep3

source_name: aerobic growth of wild-type strain on M9 media with glucose (reference)


title: anaerobic_arcAfnr_rep2


title: aerobic_wild_rep2


title: anaerobic_appY_rep1


title: aerobic_soxS_rep3

source_name: aerobic growth of soxS knock-out strain on M9 media with glucose


title: aerobic_wild_rep1


title: aerobic_oxyR_rep2

source_name: aerobic growth of oxyR knock-out strain on M9 media with glucose


title: aerobic_soxS_rep1


title: aerobic_soxS_rep2


title: aerobic_oxyR_rep1


title: aerobic_oxyR_rep3


title: aerobic_fnr_rep2

source_name: aerobic growth of fnr knock-out strain on M9 media with glucose


title: aerobic_arcA_rep1

source_name: aerobic growth of arcA knock-out strain on M9 media with glucose


title: aerobic_arcAfnr_rep3

source_name: aerobic growth of arcA/fnr double knock-out strain on M9 media with glucose


title: aerobic_arcAfnr_rep1


title: aerobic_appY_rep1

source_name: aerobic growth of appY knock-out strain on M9 media with glucose


title: aerobic_fnr_rep3


title: aerobic_fnr_rep1


title: aerobic_arcAfnr_rep2


title: aerobic_arcA_rep2


title: aerobic_appY_rep3


title: N328-2R/2R (2nd/2nd Rest CD4 T cells)

status: Public on Mar 05 2004

city: Chicago

first: Luting

last: Xu

state: IL

address: 303 E. Chicago Ave.

department: Rheumatology Division

institute: Northwestern University Feinberg School of Medicine

description:
Short-term CD4+ T cell lines were derived from human PBMC after one round of anti-CD3, anti-CD28 and IL-2 stimulation. After the first rest (1R) of 10 days, the T cells were stimulated by plate bound anti-CD3 alone (first stimulation or 1S) for 1, 3, 6, 12 or 20 hours (1S1h – 1S20h), without any costimulatory signals. After anergy induction, the T cells were rested for 3 days (second rest, 2R), and then re-stimulated with anti-CD3 for 3 or 6 hours (second stimulation - 2S3h and 2S6h). For microarrays, three groups of paired samples were studied: (i) anti-CD3-stimulated T cells from lupus patients vs. normal subjects (LN); (ii) T cells from normal subjects vs. normal subjects (NN or N1N2), to account for normal variations; and (iii) anti-CD3-stimulated normal T cells vs. normal T cells stimulated with anti-CD3 and anti-CD28 antibodies (N328).
Keywords = Lupus
Keywords = T cells

source_name: Normal CD4 T cells


title: aerobic_appY_rep2


title: N328-2S03h (2nd Stimulated CD4 T cells)


title: aerobic_arcA_rep3


title: N328-1S06h (1st Stimulated CD4 T cells)


title: N328-1S20h (1st Stimulated CD4 T cells)


title: N328-1S03h (1st Stimulated CD4 T cells)


title: N328-1S12h (1st Stimulated CD4 T cells)


title: N328-1S01h (1st Stimulated CD4 T cells)


title: N1N2-2R (2nd Rest CD4 T cells)


title: N328-2S06h (2nd Stimulated CD4 T cells)


title: N1N2-1S06h (1st Stimulated CD4 T cells)


title: N1N2-2S03h (2nd Stimulated CD4 T cells)


title: N1N2-2S06h (2nd Stimulated CD4 T cells)


title: N1N2-1S03h (1st Stimulated CD4 T cells)


title: N1N2-1S12h (1st Stimulated CD4 T cells)


title: N1N2-1S20h (1st Stimulated CD4 T cells)


title: N328-1R/2R (1st/2ndR Rest CD4 T cells)


title: N1N2-1S01h (1st Stimulated CD4 T cells)


title: LN-2R (2nd Rest CD4 T cells)

source_name: Lupus CD4 T cells


title: LN-2S06h (2nd Stimulated CD4 T cells)


title: N1N2-1R (1st Rest CD4 T cells)


title: LN-1S1h (1st Stimulated CD4 T cells)

source_name: NormalCD4 T cells


title: LN-1S20h (1st Stimulated CD4 T cells)


title: LN-1S3h (1st Stimulated CD4 T cells)


title: 35S:WIN1 WT control

status: Public on Feb 28 2004

city: York

first: Pierre

last: Broun

department: Department of Biology

country: United Kingdom

institute: CNAP - University of York

description:
RNA was extracted from pooled leaves of multiple control plants transformed with an empty binary vector using TRI Reagent according to the manufacturer’s protocol (Molecular Research Center).  Preparation of fragmented biotin-labeled cRNA, hybridizations of Arabidopsis Genome Arrays (Affymetrix 510429), washes and scanning of the slides were performed according to the manufacturer’s instructions.
Keywords = wax epidermis transcription factor

organism: Arabidopsis thaliana

source_name: Pooled leaf tissue


title: LN-1R (1st Rest CD4 T cells)


title: LN-1S6h (1st Stimulated CD4 T cells)


title: 147-630730

status: Public on Feb 27 2004

city: Ithaca

first: B. Franklin

last: Pugh

email: fp265@cornell.edu, bfp2@psu.edu

state: NY

address: 465 Biotechnology Bldg.

department: Molecular Biology and Genetics

institute: Cornell University

description:
Strains were grown in CSM-Trp-His media at 26ûC to OD600 = ~0.8. Then the cultures were shifted to 37ûC with an equal volume of heated media.  After 45 min. at 37ûC for 45 min, cells were rapidly harvested at room temperature.  Total RNA and mRNA isolation, and microarray co-hybridization of test and reference samples were performed as described (Chitikila et al., Mol. Cell, 10:871-882, 2002).
Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 4.0 analysis software.
Keywords = Saccharomyces cerevisiae

organism: Saccharomyces cerevisiae


title: LN-1S12h (1st Stimulated CD4 T cells)


title: 35S:WIN1 line 22

description:
RNA was extracted from pooled leaves of multiple T2 progenies using TRI Reagent according to the manufacturer’s protocol (Molecular Research Center).  Preparation of fragmented biotin-labeled cRNA, hybridizations of Arabidopsis Genome Arrays (Affymetrix 510429), washes and scanning of the slides were performed according to the manufacturer’s instructions.
Keywords = wax epidermis transcription factor


title: 087-660680


title: 144-650670


title: 143-680680


title: 35S:WIN1 Line 13


title: 145-600680


title: LN-2S03h (2nd Stimulated CD4 T cells)


title: 099-720680


title: 148-670670


title: 088-660680


title: 123-740680


title: 149-670670


title: 142-670690


title: 100-710680


title: 146-640650


title: 176-660660

source_name: YJS6 (CH1)

source_name: YJS9 (CH2)


title: 086-690690


title: 175-660660

source_name: YJS8 (CH2)


title: 045-730610

source_name: YJS8 (CH1)

source_name: YJS6 (CH2)


title: 193-700650

source_name: YJS9 (CH1)


title: 050-690650

source_name: YJS7 (CH2)


title: 192-680660

source_name: YJS7 (CH1)


title: 178-660660

description:
Strains were grown in CSM-Trp-His media at 26ûC to OD600 = ~0.8. The cells were rapidly harvested at room temperature.  Total RNA and mRNA isolation, and microarray co-hybridization of test and reference samples were performed as described (Chitikila et al., Mol. Cell, 10:871-882, 2002).
Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 4.0 analysis software.
Keywords = Saccharomyces cerevisiae

source_name: YJS6 not shifted to 37 (CH1)

source_name: YJS8 not shifted to 37 (CH2)


title: 048-680650

source_name: YJS8 not shifted to 37 (CH1)

source_name: YJS6 not shifted to 37 (CH2)


title: 044-660650


title: 047-660630


title: 177-660660


title: 049-660660


title: 179-680660


title: 172-680660


title: 042-680640


title: 174-680670


title: Reference vs Eosinophils

status: Public on Feb 21 2004

city: San Francisco

first: Michael

middle: E.

last: Salazar

address: 1550 Fourth Street, RM 545

institute: University of California, San Francisco

description:
Ten male 4get mice were infected with N. brasiliensis and single-cell suspensions of total lung tissue were prepared 9 days after infection.  Cells were stained for CD3, CD19, c-kit and CCR3 and sorted for CD3-CD19-c-kit-GFP+CCR3+SSChi (eosinophils) and CD3-CD19-c-kit- GFP+CCR3-SSClo (basophils) using a MoFlo high-speed cell sorter (Cytomation, Fort Collins, CO).  Total RNA was isolated from 2 x 106 eosinophils and 1.75 x 105 basophils using the Total RNA Isolation Kit (Fluka, Buchs, Switzerland) and amplified by two rounds of in vitro transcription using the Amino Allyl MessageAmpTM aRNA kit (Ambion, Austin, TX).  Universal Mouse Reference RNA (#740100; Stratagene, La Jolla, CA) was amplified in the same way.  Aminoallyl-UTP was incorporated during the second round of amplification and 5 µg of amplified RNA was coupled to Cy3 and Cy5 fluorescent dyes (CyScribeTM dye labeling kit, Amersham Biosciences, Peapack, NJ).  Probes were hybridized to spotted glass oligonucleotide (70-mer) arrays which cover just over 16,400 unique genes (Mouse Genome Set Version 2.0, Qiagen, Germany).  The arrays were prehybridized in 1% BSA (Invitrogen), 5 x SSC, 0.1% SDS for 2 hr at 42°C, washed in water and hybridized with the Cy3/Cy5-labeled probes in 2.8 x SSC, 0.2% SDS, 0.6 mg/ml Cot-1 DNA (Invitrogen), and 0.8 mg/ml yeast tRNA, at 63°C for 45 hr.  Slides were washed sequentially in 0.03%SDS/1 x SSC, 0.2 x SSC and 0.05 x SSC, dried and scanned on an Axon 4000B scanner using Genepix 3.0 software (Axon Instruments, Inc.).  Data were normalized using the R package Bioconductor software and ?lowess? normalization on the pixel medians without background subtraction (Ihaka and Gentleman, 1996).
Keywords = Eosinophils
Keywords = Basophils
Keywords = Nippostrongylus
Keywords = Lung
Keywords = Type 2 immunity

source_name: Stratagene Reference (Cy5)

source_name: Eosinophils (Cy3)


title: Reference vs Basophils

source_name: Basophils (Cy3)


title: Eosinophils vs Basophils

source_name: Eosinophils (Cy5)


title: wild type two hours exposure to zymolyase 2

status: Public on Feb 16 2004

city: Madrid

first: Javier

last: Arroyo

state: Madrid

address: Plaza Ramon y Cajal S/N

department: Microbiologia II

country: Spain

institute: Facultad de Farmacia (UCM)

description: S. cerevisiae was grown on YEPD. For Zymolyase experiments, yeast cells were grown overnight at 24 °C to an optical density 0.8 - 1 (A600). The culture was refreshed to 0.2 O.D and grown at 24 °C for 2h 30min. Next, culture was divided into two parts. One continues growing under same conditions (non-treated culture) while the other was supplemented with 5units/ml Zymolyase 100T . Cells were collected at 2 hours of growth, frozen at -80 °C and processed for RNA extraction.

source_name: BY4741 Strain (2 hours zymolyase treatment)

source_name: BY4741 Strain (Control mRNA: no treatment)


title: DD035.033 Sep 24 Atlantic Salmon Head Kidney Study Slide 033

status: Public on Feb 11 2004

city: Milwaukee

first: Matthew

middle: Lawrence

last: Rise

state: WI

address: 600 E. Greenfield Ave.

department: Great Lakes WATER Institute

institute: University of Wisconsin - Milwaukee

description:
MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon head kidney. Head kidney mRNA samples were prepared from pooled (n = 10) infected or control kidney. Approximately 200 ng of infected or control head kidney mRNA was used as template in aRNA synthesis reactions. Two infected head kidney samples were pooled to yield 8.9 ug of aRNA, and a single control head kidney sample contained 8.4 ug of aRNA. Infected and control head kidney aRNA samples were visualized on agarose gels to check quality and quantity (data not shown). Except for the amounts of aRNA and reverse transcription (RT) primer used, head kidney target labeling reactions and microarray hybridizations were identical. Head kidney target syntheses used 2 ug of aRNA and 2 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C for 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5’T18[Q-N]3’), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before.
Keywords = Atlantic salmon
Keywords = rainbow trout
Keywords = salmonid
Keywords = rickettsia
Keywords = head kidney
Keywords = hematopoietic kidney
Lot batch = DD035

organism: Salmo salar

source_name: Cy3-Labeled Non-infected Salmo salar head kidney

source_name: Cy5-Labeled Piscirickettsia salmonis-infected Salmo salar head kidney


title: BLI_20855

status: Public on Feb 07 2004

city: Tsukuba City, Ibaraki

first: Chandana

last: Herath

address: Ikenodai 2

department: Development Biology

institute: National Institute of Agrobiological Sciences

description: Ten-microgram of liver total RNA obtained from nonpregnant cycling cows was reverse transcribed in the presence of cyanine-3-fluorescent dye (Cy3)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy3 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained from bovine NT calf was separately reverse transcribed in the presence of Cy5-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy5 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. A labeled control cDNA probe and a test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The ‘arrayIt’ hybridization cassette chamber (arrayIt, TeleChem International, Inc., Sunnyvale, CA) and lid were rinsed with autoclaved MilliQ water and dried thoroughly with paper towel. Twenty microliter of autoclaved MilliQ was placed in both grooves on either side of the cassette to maintain high humidity in the cassette chamber during hybridization. Then the microarray slide, which had been pretreated by passing through various solutions was inserted with the spotted cDNA side facing upwards. The labeled probe mixture was then applied to the microarray and placed a cover slip carefully and covered with the cassette lid. The cassette chamber was incubated in a water bath preheated to 65oC for 12 – 15 h. After incubation for 12 – 15 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed. This was followed by immediate scanning and image acquisition.

organism: Bos taurus

source_name: Fetal liver-AI (day 150 of pregnancy)

source_name: Adult nonpregnant liver


title: DD035.032 Sep 24 Atlantic Salmon Head Kidney Study Slide 032

description:
MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon head kidney. Head kidney mRNA samples were prepared from pooled (n = 10) infected or control kidney. Approximately 200 ng of infected or control head kidney mRNA was used as template in aRNA synthesis reactions. Two infected head kidney samples were pooled to yield 8.9 ug of aRNA, and a single control head kidney sample contained 8.4 ug of aRNA. Infected and control head kidney aRNA samples were visualized on agarose gels to check quality and quantity (data not shown). Except for the amounts of aRNA and reverse transcription (RT) primer used, head kidney target labeling reactions and microarray hybridizations were identical. Head kidney target syntheses used 2 ug of aRNA and 2 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C for 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5’T18[Q-N]3’), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before.

Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 75 Cy3, PMT 63 or 64 Cy5 for head kidney study). Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery).

Keywords = Atlantic salmon
Keywords = rainbow trout
Keywords = salmonid
Keywords = rickettsia
Keywords = head kidney
Keywords = hematopoietic kidney
Lot batch = DD035

source_name: Cy3-Labeled Piscirickettsia salmonis-infected Salmo salar head kidney

source_name: Cy5-Labeled Non-infected Salmo salar head kidney


title: DD035.034 Sep 24 Atlantic Salmon Head Kidney Study Slide 034


title: CL012.003 Feb 12 Atlantic Salmon Macrophage Study Slide 003

description:
MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon macrophages.  Infected and control (24 h incubation) macrophage mRNA samples were subjected to two rounds of amplification using the MessageAmp aRNA kit (Ambion) and manufacturer’s instructions.  Approximately 400 ng of macrophage mRNA was used as template in the first round of amplification, yielding 8.1 ug of infected macrophage aRNA and 7.8 ug of control macrophage aRNA.  Approximately 1 ug of first-round macrophage aRNA was used as template in the second round of amplification, yielding 81.2 ug of infected macrophage aRNA and 83.3 ug of control macrophage aRNA.  Infected and control macrophage aRNA samples were visualized on agarose gels to check quality and quantity.  Except for the amounts of aRNA and reverse transcription (RT) primer used, macrophage target labeling reactions and microarray hybridizations were identical.  Macrophage target syntheses used 5 ug of second-round aRNA and 5 ug of random primers (Roche).  RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume.  Samples were incubated at 70 degrees C 4 min, then placed on ice.  RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen).  RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min.  Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight.  Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer:  50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5’T18[Q-N]3’), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma).  Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays.  Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube).  Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h.  Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC.  Slides were dried by centrifugation as before.

Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer).  The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 72 Cy3, PMT 63 or 64 Cy5 for macrophage study).  Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery).
Keywords = Atlantic salmon
Keywords = rainbow trout
Keywords = salmonid
Keywords = rickettsia
Keywords = macrophage
Lot batch = CL012

source_name: Cy3 24-hour Piscirickettsia salmonis-infected Salmo salar macrophage

source_name: Cy5 24-hour non-infected Salmo salar macrophage


title: CL012.005 Feb 12 Atlantic Salmon Macrophage Study Slide 005

description:
MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon macrophages. Infected and control (24 h incubation) macrophage mRNA samples were subjected to two rounds of amplification using the MessageAmp aRNA kit (Ambion) and manufacturer’s instructions. Approximately 400 ng of macrophage mRNA was used as template in the first round of amplification, yielding 8.1 ug of infected macrophage aRNA and 7.8 ug of control macrophage aRNA. Approximately 1 ug of first-round macrophage aRNA was used as template in the second round of amplification, yielding 81.2 ug of infected macrophage aRNA and 83.3 ug of control macrophage aRNA. Infected and control macrophage aRNA samples were visualized on agarose gels to check quality and quantity. Except for the amounts of aRNA and reverse transcription (RT) primer used, macrophage target labeling reactions and microarray hybridizations were identical. Macrophage target syntheses used 5 ug of second-round aRNA and 5 ug of random primers (Roche). RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume. Samples were incubated at 70 degrees C 4 min, then placed on ice. RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen). RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min. Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight. Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer: 50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5’T18[Q-N]3’), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma). Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays. Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube). Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h. Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC. Slides were dried by centrifugation as before.

Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer). The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 72 Cy3, PMT 63 or 64 Cy5 for macrophage study). Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery).
Keywords = Atlantic salmon
Keywords = rainbow trout
Keywords = salmonid
Keywords = rickettsia
Keywords = macrophage
Lot batch = CL012

source_name: Cy3 24-hour non-infected Salmo salar macrophage

source_name: Cy5 24-hour Piscirickettsia salmonis-infected Salmo salar macrophage


title: wild type two hours exposure to zymolyase 4


title: wild type two hours exposure to zymolyase 3


title: BLI_20847

description: Ten-microgram of liver total RNA obtained from normal fetaus was reverse transcribed in the presence of cyanine-3-fluorescent dye (Cy3)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy3 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained from bovine NT fetus was separately reverse transcribed in the presence of Cy5-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy5 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. A labeled control cDNA probe and a test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The ‘arrayIt’ hybridization cassette chamber (arrayIt, TeleChem International, Inc., Sunnyvale, CA) and lid were rinsed with autoclaved MilliQ water and dried thoroughly with paper towel. Twenty microliter of autoclaved MilliQ was placed in both grooves on either side of the cassette to maintain high humidity in the cassette chamber during hybridization. Then the microarray slide, which had been pretreated by passing through various solutions was inserted with the spotted cDNA side facing upwards. The labeled probe mixture was then applied to the microarray and placed a cover slip carefully and covered with the cassette lid. The cassette chamber was incubated in a water bath preheated to 65oC for 12 – 15 h. After incubation for 12 – 15 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed. This was followed by immediate scanning and image acquisition.

source_name: Fetal liver-NT#1 (day48)

source_name: Fetal liver-IVF (day54)


title: CL012.004 Feb 12 Atlantic Salmon Macrophage Study Slide 004


title: RatLung_Sh_1

city: San Antonio

first: Phillip

last: Bowman

address: 3400 Rawley E. Chambers Ave

institute: Institute of Surgical Research

description:
Male Sprague-Dawley rats weighing 280-300 g were anesthetized with 1.75% to 2.5% isofluorane and instrumented with two arterial catheters, one externalized for blood withdrawal and the other (not externalized) attached to the Data Sciences International (DSI) PhysioTel (St. Paul, MN, model C50-PXT) monitoring device placed intraperitoneally and used in monitoring blood pressure, temperature, and electrocardiogram data via telemetry.  After 7 to 10 days of recovery, and only after the animals had begun to gain weight, the experiment was performed.  At the beginning of hemorrhage (H), blood was withdrawn manually through the externalized catheter with 1-ml syringes at l ml/min.  Total blood volume was estimated to be 6.5% of the body weight and 40% of this (2.6 ml/100g) was removed to effect the hemorrhage.
Control animals (Con) were uninstrumented and were not hemorrhaged or manipulated in any way. Sham (Sh) rats were instrumented, but not hemorrhaged, and physiologic measures were taken for 4 – 23 hrs 6 hrs prior to euthanasia. At 1, 3, 6, 16, 24, 48, or 72 hours after hemorrhage animals were deeply anesthetized with Nembutal (50 mg/kg), and the post hemorrhage blood sample was collected by cardiac puncture and the animals were euthanized by exsanguination.  All tissues were collected within 10 minutes of death and 100 mg amounts of the following tissues were removed and placed in RNALater™  (Ambion, Austin, TX):  intestine, liver, lung, kidney, spleen, heart, skeletal muscle (gastrocnemius) skin, and brain.
Total RNA was isolated from each tissue from each animal with 2 rounds of TRI Reagent® (MRC, Cincinnati, OH) and its quality analyzed by gel electrophoresis.  A reference preparation consisting of equal amounts of RNA pooled from 10 organs (liver, lung, kidney, spleen, heart, skeletal muscle, skin, jejunum, and brain) of untreated control animals was used as reference RNA.  Five-µg samples exhibiting undegraded RNA from each rat lung were reverse transcribed with Superscript II™ (Invitrogen Corp., Carlsbad, CA) in the presence of a C-6 amine modified random hexamer and aminoallodeoxyuridine to produce fluorescent labeled cDNA.  RNA from the reference preparation was all labeled together.  Following reverse transcription, RNA was degraded with 0.1 M NaOH and neutralized with ph 7.0 Hepes buffer.  Following isolation on Qiaquick columns (Qiagen), elution and lyophilization, the lung-sample was labeled with Cy5 ester and reference cDNA samples with Cy3 ester (Amersham Biotech, Piscataway, NJ) After separation from unbound dye, the samples were again lyophilized and then reconstituted in hybridization solution (32% formamide, 5 X Denhardt's solutions, 3X SSC, 20 mM Hepes buffer, ph 7.0)  for 20 hrs at 42 degree. Following hybridization on a microarray (one per rat) and washing to remove unhybridized cDNA and scanning with an Axon 4000B (Axon Instruments, Union City, CA) at 10 micron resolution, the resulting 16-bit TIFF images were analyzed with GenePix 4.1 software (Axon Instruments) for calculation of Cy5 and Cy3 fluorescence intensities at each spot.
Keywords = rat lung hemorrhage

source_name: Lung of sham animal

source_name: Pooled RNA of equal amounts from 10 organs (liver, lung, kidney, spleen, heart, skeletal muscle, skin, jejunum, and brain) of untreated control animals


title: BLI_20852

description: Ten-microgram of liver total RNA obtained from normal calf was reverse transcribed in the presence of cyanine-3-fluorescent dye (Cy3)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy3 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained from bovine NT calf was separately reverse transcribed in the presence of Cy5-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy5 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. A labeled control cDNA probe and a test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The ‘arrayIt’ hybridization cassette chamber (arrayIt, TeleChem International, Inc., Sunnyvale, CA) and lid were rinsed with autoclaved MilliQ water and dried thoroughly with paper towel. Twenty microliter of autoclaved MilliQ was placed in both grooves on either side of the cassette to maintain high humidity in the cassette chamber during hybridization. Then the microarray slide, which had been pretreated by passing through various solutions was inserted with the spotted cDNA side facing upwards. The labeled probe mixture was then applied to the microarray and placed a cover slip carefully and covered with the cassette lid. The cassette chamber was incubated in a water bath preheated to 65oC for 12 – 15 h. After incubation for 12 – 15 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed. This was followed by immediate scanning and image acquisition.

source_name: Calf liver-NT (day13)

source_name: Calf liver-AI (day13)


title: wild type two hours exposure to zymolyase 1

source_name: BY4741  Strain (Control mRNA: no treatment)

source_name: BY4741  Strain (2 hours zymolyase treatment)


title: BLI_20854

description: Ten-microgram of liver total RNA obtained from normal calf was reverse transcribed in the presence of cyanine-5-fluorescent dye (Cy5)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy5 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained from bovine NT calf was separately reverse transcribed in the presence of Cy3-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy3 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. A labeled control cDNA probe and a test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The ‘arrayIt’ hybridization cassette chamber (arrayIt, TeleChem International, Inc., Sunnyvale, CA) and lid were rinsed with autoclaved MilliQ water and dried thoroughly with paper towel. Twenty microliter of autoclaved MilliQ was placed in both grooves on either side of the cassette to maintain high humidity in the cassette chamber during hybridization. Then the microarray slide, which had been pretreated by passing through various solutions was inserted with the spotted cDNA side facing upwards. The labeled probe mixture was then applied to the microarray and placed a cover slip carefully and covered with the cassette lid. The cassette chamber was incubated in a water bath preheated to 65oC for 12 – 15 h. After incubation for 12 – 15 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed. This was followed by immediate scanning and image acquisition.


title: DD035.031 Sep 24 Atlantic Salmon Head Kidney Study Slide 031

description:
MicroPoly(A)Pure kits (Ambion) were used to prepare mRNA from P. salmonis-infected and control Atlantic salmon head kidney.  Head kidney mRNA samples were prepared from pooled (n = 10) infected or control kidney.  Approximately 200 ng of infected or control head kidney mRNA was used as template in aRNA synthesis reactions.  Two infected head kidney samples were pooled to yield 8.9 ug of aRNA, and a single control head kidney sample contained 8.4 ug of aRNA.  Infected and control head kidney aRNA samples were visualized on agarose gels to check quality and quantity (data not shown).  Except for the amounts of aRNA and reverse transcription (RT) primer used, head kidney target labeling reactions and microarray hybridizations were identical.  Head kidney target syntheses used 2 ug of aRNA and 2 ug of random primers (Roche).  RT primers were mixed with aRNA samples, and nuclease-free H2O (Invitrogen) was added to make 10 ul total volume.  Samples were incubated at 70 degrees C for 4 min, then placed on ice.  RT reactions included 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM each dATP, dCTP, and dGTP, 0.2 mM dTTP, 0.05 mM Cy3-dUTP or Cy5-dUTP (Amersham), 40U RNAguard ribonuclease inhibitor (Amersham), and 400U Superscript II Rnase H- reverse transcriptase (Invitrogen).  RT reactions were incubated at 42 degrees C for 2.5 h, followed by addition of 0.025U RNase A (Sigma) and 1.5U RNase H (New England BioLabs) and incubation at 37 degrees C for 30 min.  Labeled targets were cleaned using the Qiaquick PCR purification kit (Qiagen) and precipitated, with 300 mM sodium acetate, 1 ul of 20 ug/ul glycogen (Invitrogen), and 2.5 volumes of 100% ethanol, at -20 degrees C overnight.  Each labeled target was recovered by centrifugation (14,000 rpm, 4 degrees C, 1 h), washed in 70% ethanol, air-dried, and resuspended in 60 ul of hybridization buffer:  50% deionized formamide, 5X SSC, 0.1% SDS, 5 ul of 5 ug/ul oligo dT (5’T18[Q-N]3’), 5 ul of 2 ug/ul BSA (Pierce), and 2 ul of 10 ug/ul sonicated human placental DNA (Sigma).  Targets were incubated at 96 degrees C for 3 min, and then at 65 degrees C until applied to microarrays.  Microarrays were prepared for hybridization by washing 2 X 5 min in 0.1% SDS, washing 5 X 1 min in MilliQ H2O, immersing 3 min in 95 degrees C MilliQ H2O, and drying by centrifugation (2000 rpm, 5 min, in 50 ml conical tube).  Microarray hybridizations were run in the dark under HybriSlips hybridization covers (Grace Biolabs) in slide hybridization chambers (Corning) submerged in a 45 degrees C water bath for 16 h.  Coverslips were floated off in 45 degrees C (1X SSC, 0.2% SDS), and arrays were washed 1 X 10 min in 45 degrees C (1X SSC, 0.2% SDS), 3 X 4 min in room temperature (0.1X SSC, 0.1% SDS), and 4 X 4 min in room temperature 0.1X SSC.  Slides were dried by centrifugation as before.

Fluorescent images of hybridized arrays were acquired immediately at 10 um resolution using ScanArray Express (PerkinElmer).  The Cy3 and Cy5 cyanine fluors were excited at 543 nm and 633 nm respectively, and the same laser power (90%) and photomultiplier tube (PMT) settings were used for all slides in a study (PMT 75 Cy3, PMT 63 or 64 Cy5 for head kidney study).  Fluorescent intensity data were extracted from TIF images using Imagene 5.5 software (Biodiscovery).

Keywords = Atlantic salmon
Keywords = rainbow trout
Keywords = salmonid
Keywords = rickettsia
Keywords = head kidney
Keywords = hematopoietic kidney
Lot batch = DD035


title: BLI_20853


title: BLI_20842

description: Ten-microgram of pooled liver total RNA obtained from nonpregnant cycling cows was reverse transcribed in the presence of cyanine-3-fluorescent dye (Cy3)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy3 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained from bovine fetal liver was separately reverse transcribed in the presence of Cy5-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy5 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. A labeled control cDNA probe and a test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The ‘arrayIt’ hybridization cassette chamber (arrayIt, TeleChem International, Inc., Sunnyvale, CA) and lid were rinsed with autoclaved MilliQ water and dried thoroughly with paper towel. Twenty microliter of autoclaved MilliQ was placed in both grooves on either side of the cassette to maintain high humidity in the cassette chamber during hybridization. Then the microarray slide, which had been pretreated by passing through various solutions was inserted with the spotted cDNA side facing upwards. The labeled probe mixture was then applied to the microarray and placed a cover slip carefully and covered with the cassette lid. The cassette chamber was incubated in a water bath preheated to 65oC for 12 – 15 h. After incubation for 12 – 15 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed. This was followed by immediate scanning and image acquisition.

source_name: Fetal liver-AI (day 65 of pregnancy)


title: BLI_20835

source_name: Fetal liver-NT#2 (day 48 of pregnancy)


title: BLI_20841

description: Ten-microgram of pooled liver total RNA obtained from nonpregnant cycling cows was reverse transcribed in the presence of cyanine-5-fluorescent dye (Cy5)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy5 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained from bovine fetal liver was separately reverse transcribed in the presence of Cy3-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy3 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. A labeled control cDNA probe and a test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The ‘arrayIt’ hybridization cassette chamber (arrayIt, TeleChem International, Inc., Sunnyvale, CA) and lid were rinsed with autoclaved MilliQ water and dried thoroughly with paper towel. Twenty microliter of autoclaved MilliQ was placed in both grooves on either side of the cassette to maintain high humidity in the cassette chamber during hybridization. Then the microarray slide, which had been pretreated by passing through various solutions was inserted with the spotted cDNA side facing upwards. The labeled probe mixture was then applied to the microarray and placed a cover slip carefully and covered with the cassette lid. The cassette chamber was incubated in a water bath preheated to 65oC for 12 – 15 h. After incubation for 12 – 15 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed. This was followed by immediate scanning and image acquisition.

source_name: lt nonpregnant liver


title: Duchenne muscular dystrophy (DMD) patient’s skeletal muscle sample 12

status: Public on Feb 04 2004

first: Judith

last: Haslett

institute: Harvard Medical School

description:
biopsy muscle sample obtained from a 3-year-old male DMD patient
Keywords = gene expression profile of DMD pateint's skeletal muscle
Keywords = Affymetrix HG-U95B

source_name: Quadriceps


title: BLI_20837

source_name: Fetal liver-IVF (day 54 of pregnancy)

source_name: Adult nonpregnant liver


title: BLI_20836


title: Duchenne muscular dystrophy (DMD) patient’s skeletal muscle sample 8

description:
biopsy muscle sample obtained from a 0.8-year-old male DMD patient
Keywords = gene expression profile of DMD pateint's skeletal muscle
Keywords = Affymetrix HG-U95B


title: BLI_20845


title: Duchenne muscular dystrophy (DMD) patient’s skeletal muscle sample 5

description:
biopsy muscle sample obtained from a 3.5-year-old male DMD patient
Keywords = gene expression profile of DMD pateint's skeletal muscle
Keywords = Affymetrix HG-U95B


title: BLI_20846

description: Ten-microgram of liver total RNA obtained from normal fetal liver was reverse transcribed in the presence of cyanine-5-fluorescent dye (Cy5)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy5 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained from bovine NT fetus was separately reverse transcribed in the presence of Cy3-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy3 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. A labeled control cDNA probe and a test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The ‘arrayIt’ hybridization cassette chamber (arrayIt, TeleChem International, Inc., Sunnyvale, CA) and lid were rinsed with autoclaved MilliQ water and dried thoroughly with paper towel. Twenty microliter of autoclaved MilliQ was placed in both grooves on either side of the cassette to maintain high humidity in the cassette chamber during hybridization. Then the microarray slide, which had been pretreated by passing through various solutions was inserted with the spotted cDNA side facing upwards. The labeled probe mixture was then applied to the microarray and placed a cover slip carefully and covered with the cassette lid. The cassette chamber was incubated in a water bath preheated to 65oC for 12 – 15 h. After incubation for 12 – 15 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed. This was followed by immediate scanning and image acquisition.


title: Duchenne muscular dystrophy (DMD) patient’s skeletal muscle sample 7


title: BLI_20833

source_name: Fetal liver-NT#3 (day 56 of pregnancy)


title: BLI_20831

source_name: Fetal liver-AI (day 245 of pregnancy-sample#2)


title: Duchenne muscular dystrophy (DMD) patient’s skeletal muscle sample 6

description:
biopsy muscle sample obtained from a 1-year-old male DMD patient
Keywords = gene expression profile of DMD pateint's skeletal muscle
Keywords = Affymetrix HG-U95B


title: BLI_20832


title: BLI_20826

source_name: Fetal liver-AI (day 245 of pregnancy)


title: BLI_20825


title: Duchenne muscular dystrophy (DMD) patient’s skeletal muscle sample 11

description:
biopsy muscle sample obtained from a 4-year-old male DMD patient
Keywords = gene expression profile of DMD pateint's skeletal muscle
Keywords = Affymetrix HG-U95B


title: BLI_20834


title: Duchenne muscular dystrophy (DMD) patient’s skeletal muscle sample 2

description:
biopsy muscle sample obtained from a 1.5-year-old male DMD patient
Keywords = gene expression profile of DMD pateint's skeletal muscle
Keywords = Affymetrix HG-U95B


title: BLI_20823

source_name: Fetal liver-IVF (day 135 of pregnancy)


title: BLI_20824


title: Duchenne muscular dystrophy (DMD) patient’s skeletal muscle sample 1


title: Duchenne muscular dystrophy (DMD) patient’s skeletal muscle sample 3

description:
biopsy muscle sample obtained from a 7-year-old male DMD patient
Keywords = gene expression profile of DMD pateint's skeletal muscle
Keywords = Affymetrix HG-U95B


title: BLI_20827


title: Duchenne muscular dystrophy (DMD) patient’s skeletal muscle sample 4


title: BLI_20813

description: Ten-microgram of pooled liver total RNA obtained from nonpregnant cycling cows was reverse transcribed in the presence of cyanine-5-fluorescent dye (Cy5)-conjugated dUTP (Amersham Pharmacia Biotech) by using Superscript II reverse transcriptase (Life Technologies, Rockville, MD) to yield Cy5 fluorescence-labeled control cDNA probe. Similarly, liver total RNA obtained from the bovine NT-fetal liver was separately reverse transcribed in the presence of Cy3-conjugated dUTP by using Superscript II reverse transcriptase to yield Cy3 fluorescence-labeled test cDNA probe. After a 2-h reverse transcription reaction at 42oC, RNA was degraded by addition of 1.5 µl of an alkaline solution (1N NaOH / 20 mM EDTA), and neutralized by addition of 1.5 µl 1N HCl. A labeled control cDNA probe and a test cDNA probe were then mixed and washed by centrifugation with TE (Tris-EDTA, pH 7.0) first and then with TE, 20 µg Salmon sperm DNA, 20 µg polyA and 20 µg yeast RNA in a Microcon YM-30 column (Millipore, Bedford, MA). The concentrated probe mix was adjusted with TE to a volume of 24.8 µl, denatured at 100oC for 2 min, spun for a few min and made to a final hybridization volume of 31 µl with 5.27 µl 20XSSC and 0.93 µl 10% SDS and kept at room temperature until used. Before hybridization, microarray glass slides were immersed in 0.2% SDS for 2 min followed by two washes each 2 min in autoclaved Milli Q water, and subjected to blocking in 180 ml of Methyl Pyrrolidinone (Aldrich) containing 2.7 g Succinic Anhydride (WAKO, Japan) and 15.3 ml 1M Boric acid (pH 8.0) solution for 20 min. Slides were then washed with autoclaved Milli Q water, and the spotted cDNA targets (microarray) were denatured in boiling water for 2 min. The slides were dehydrated in 100% ethanol for 2 min and air dried by spinning at a low speed. The ‘arrayIt’ hybridization cassette chamber (arrayIt, TeleChem International, Inc., Sunnyvale, CA) and lid were rinsed with autoclaved MilliQ water and dried thoroughly with paper towel. Twenty microliter of autoclaved MilliQ was placed in both grooves on either side of the cassette to maintain high humidity in the cassette chamber during hybridization. Then the microarray slide, which had been pretreated by passing through various solutions was inserted with the spotted cDNA side facing upwards. The labeled probe mixture was then applied to the microarray and placed a cover slip carefully and covered with the cassette lid. The cassette chamber was incubated in a water bath preheated to 65oC for 12 – 15 h. After incubation for 12 – 15 h at 65oC, the slides were washed in 2 x SSC / 0.03% SDS for 2 min, followed by washing in 2 x SSC, 1 x SSC and 0.2 x SSC, each for 2 min with gentle agitation, and air dried by spinning at a low speed. This was followed by immediate scanning and image acquisition.

source_name: Fetal liver-NT#1 (day48)


title: duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 12

description:
biopsy muscle sample obtained from a 3-year-old duchenne muscular dystrophy male patient
Keywords = Gene expression profiles of duchenne muscular dystrophy patient's skeletal muscle sample
Keywords = Affymetrix HG-U95E


title: Duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 10(repeat)

description:
biopsy muscle sample obtained from a duchenne muscular dystrophy male patient (Age: unknown)
Keywords = Gene expression profiles of DMD patient's skeletal muscle sample
Keywords = Affymetrix HG-U95D


title: duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 8

description:
biopsy muscle sample obtained from a 6-year-old duchenne muscular dystrophy male patient
Keywords = Gene expression profiles of duchenne muscular dystrophy patient's skeletal muscle sample
Keywords = Affymetrix HG-U95E


title: Duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 6(repeat)

description:
biopsy muscle sample obtained from a 1-year-old duchenne muscular dystrophy male patient
Keywords = Gene expression profiles of DMD patient's skeletal muscle sample
Keywords = Affymetrix HG-U95D


title: duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 11

description:
biopsy muscle sample obtained from a 4-year-old duchenne muscular dystrophy male patient
Keywords = Gene expression profiles of duchenne muscular dystrophy patient's skeletal muscle sample
Keywords = Affymetrix HG-U95E


title: duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 7

description:
biopsy muscle sample obtained from a 0.8-year-old duchenne muscular dystrophy male patient
Keywords = Gene expression profiles of duchenne muscular dystrophy patient's skeletal muscle sample
Keywords = Affymetrix HG-U95E


title: Duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 11

description:
biopsy muscle sample obtained from a 4-year-old duchenne muscular dystrophy male patient
Keywords = Gene expression profiles of DMD patient's skeletal muscle sample
Keywords = Affymetrix HG-U95D


title: duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 2

description:
biopsy muscle sample obtained from a 1.5-year-old duchenne muscular dystrophy male patient
Keywords = Gene expression profiles of duchenne muscular dystrophy patient's skeletal muscle sample
Keywords = Affymetrix HG-U95E


title: duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 6

description:
biopsy muscle sample obtained from a 1-year-old duchenne muscular dystrophy male patient
Keywords = Gene expression profiles of duchenne muscular dystrophy patient's skeletal muscle sample
Keywords = Affymetrix HG-U95E


title: Duchenne Muscular dystrophy (DMD) patient’s skeletal muscle sample 12 (repeat)

description:
biopsy muscle sample obtained from a 3-year-old male individual
Keywords = Gene expression profiles of duchenne muscular dystrophy patient's skeletal muscle sample
Keywords = Affymetrix HG-U95C


title: Duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 1


title: duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 3

description:
biopsy muscle sample obtained from a 7-year-old duchenne muscular dystrophy male patient
Keywords = Gene expression profiles of duchenne muscular dystrophy patient's skeletal muscle sample
Keywords = Affymetrix HG-U95E


title: duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 1(repeat)

description:
biopsy muscle sample obtained from a 1-year-old male individual
Keywords = Gene expression profiles of duchenne muscular dystrophy patient's skeletal muscle sample
Keywords = Affymetrix HG-U95E


title: Duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 2

description:
biopsy muscle sample obtained from a 1.5-year-old duchenne muscular dystrophy male patient
Keywords = Gene expression profiles of DMD patient's skeletal muscle sample
Keywords = Affymetrix HG-U95D


title: Duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 6


title: Duchenne Muscular dystrophy (DMD) patient’s skeletal muscle sample 7

description:
biopsy muscle sample obtained from a 0.8-year-old male individual
Keywords = Gene expression profiles of duchenne muscular dystrophy patient's skeletal muscle sample
Keywords = Affymetrix HG-U95C


title: Duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 4

description:
biopsy muscle sample obtained from a 3-year-old duchenne muscular dystrophy male patient
Keywords = Gene expression profiles of DMD patient's skeletal muscle sample
Keywords = Affymetrix HG-U95D


title: Duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 8

description:
biopsy muscle sample obtained from a 6-year-old duchenne muscular dystrophy male patient
Keywords = Gene expression profiles of DMD patient's skeletal muscle sample
Keywords = Affymetrix HG-U95D


title: Duchenne Muscular dystrophy (DMD) patient’s skeletal muscle sample 12


title: duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 4


title: Duchenne Muscular dystrophy (DMD) patient’s skeletal muscle sample 8

description:
biopsy muscle sample obtained from a 6-year-old male individual
Keywords = Gene expression profiles of duchenne muscular dystrophy patient's skeletal muscle sample
Keywords = Affymetrix HG-U95C


title: Duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 10

description:
biopsy muscle sample obtained from a duchenne muscular dystrophy male patient( age: unknown)
Keywords = Gene expression profiles of DMD patient's skeletal muscle sample
Keywords = Affymetrix HG-U95D


title: Duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 9


title: duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 1


title: Duchenne Muscular dystrophy (DMD) patient’s skeletal muscle sample 9

description:
biopsy muscle sample obtained from a  male individual (age: unknown)
Keywords = Gene expression profiles of duchenne muscular dystrophy patient's skeletal muscle sample
Keywords = Affymetrix HG-U95C


title: Duchenne Muscular dystrophy (DMD) patient’s skeletal muscle sample 11

description:
biopsy muscle sample obtained from a  4-year-old male individual
Keywords = Gene expression profiles of duchenne muscular dystrophy patient's skeletal muscle sample
Keywords = Affymetrix HG-U95C


title: Duchenne Muscular Dystrophy (DMD) patient’s skeletal muscle sample 7

description:
biopsy muscle sample obtained from a 0.8-year-old duchenne muscular dystrophy male patient
Keywords = Gene expression profiles of DMD patient's skeletal muscle sample
Keywords = Affymetrix HG-U95D


title: Duchenne Muscular dystrophy (DMD) patient’s skeletal muscle sample 3

description:
biopsy muscle sample obtained from a 7-year-old male individual
Keywords = Gene expression profiles of duchenne muscular dystrophy patient's skeletal muscle sample
Keywords = Affymetrix HG-U95C


title: Duchenne Muscular dystrophy (DMD) patient’s skeletal muscle sample 4


title: Duchenne Muscular dystrophy (DMD) patient’s skeletal muscle sample 6

description:
biopsy muscle sample obtained from a 1-year-old male individual
Keywords = Gene expression profiles of duchenne muscular dystrophy patient's skeletal muscle sample
Keywords = Affymetrix HG-U95C


title: Duchenne Muscular dystrophy (DMD) patient’s skeletal muscle sample 2

description:
biopsy muscle sample obtained from a 1.5-year-old male individual
Keywords = Gene expression profiles of duchenne muscular dystrophy patient's skeletal muscle sample
Keywords = Affymetrix HG-U95C


title: Duchenne Muscular dystrophy (DMD) patient’s skeletal muscle sample 1

description:
biopsy muscle sample obtained from a 1-year-old male individual
Keywords = Gene expression profiles of duchennemuscular dystrophy patient's skeletal muscle sample
Keywords = Affymetrix HG-U95C


title: Amino Terminated surface 32 hours

status: Public on Jan 28 2004

first: Catherine

last: Klapperich

address: 44 Cummington St. 7th Floor

department: Biomedical and Manufacturing Engineering

description: Cultured in DMEM with 10% FBS and penicillin (100U/ml)/streptomycin (100ug/ml). Media changed every other day. 37ºC, 5% CO2.

source_name: Human Osteosarcoma TE85 cells


title: d3 heads, paraquat-exposed

status: Public on Jan 18 2004

first: Sige

last: Zou

department: Physiology Department

description:
To profile the effect of oxidative stress on gene expression in Drosophila, w118 male adult flies were cultured.  Adult males were collected within 24 hr after eclosion.  Approximately 200 flies were maintained in constant darkness in each food bottle at 25°C and 70% humidity.  At adult d2, the flies were fed sucrose with 15mM paraquat (experimental condition) or regular sucrose (control condition) for 30 hours.  Heads were collected and transcripts extracted as described above, then profiled using Affymetrix GeneChips.

To separate the head from the rest of the body, flies were frozen and briefly vortexed in liquid nitrogen. Fly heads were collected using a sieve which retained fly bodies.  Total RNA was extracted using Trizol (GIBCO/BRL).  Poly(A) RNA was isolated using Oligotex resin (Qiagen).  Samples were profiled with Affymetrix DrosGenome1 GeneChips, using standard Affymetrix protocol.

organism: Drosophila melanogaster

source_name: Heads from adult d3 males, w118 strain, after 30hr paraquat exposure


title: Tissue Culture Polystyrene 6 hours

description:
Cultured in DMEM with 10% FBS and penicillin (100U/ml)/streptomycin (100ug/ml).
Media changed every other day.
37ºC, 5% CO2.
Cells plated on sample surfaces.


title: d3 heads

description:
The w1118 strain is an inbred lab stock widely used in genetic and transgenic studies.  w1118 males have a median life span of 35 days.

Adult males were collected within 24 hr after eclosion.  Approximately 200 flies were maintained in constant darkness in each food bottle at 25°C and 70% humidity and were transferred to fresh bottles every 3-4 days.   Transcripts were harvested at d3 and d47.

To separate the head from the rest of the body, flies were frozen and briefly vortexed in liquid nitrogen. Fly heads were collected using a sieve which retained fly bodies.  Total RNA was extracted using Trizol (GIBCO/BRL).  Poly(A) RNA was isolated using Oligotex resin (Qiagen).  Samples were profiled with Affymetrix DrosGenome1 GeneChips, using standard Affymetrix protocol.

source_name: Heads from adult d3 males, w118 strain


title: d47 heads

source_name: Heads from adult d47 males, w118 strain


title: Tissue Culture Polystyrene 32 hours


title: N-6_PRE

status: Public on Jan 17 2004

city: Minneapolis

first: Grindle

middle: M

last: Suzanne

state: MN

address: Dwan CVRC 289, MMC 508, 420 Delaware Street SE

department: Division of Cardiology

institute: Lillehei Heart Institute

description:
19 paired human left ventricular apex samples were harvested at the time of implant of a left ventricular assist device (PRE) and at the time of explant (POST). The cohort included patients that were clinically classified as "ischemic" (I) showing evidence of coronary artery disease, "non-ischemic" (N) no evidence of coronary artery disease or "acute Myocardial  infarction" (IM) myocardial infarction within 10 days of the implant. Tissue was processed and hybridized to the Affymetrix HG-U133A chip.
Keywords = Heart, Cardiac, Assist Device, LVAD, GATA-4, Sprouty1, microarray, heart failure

source_name: Left Cardiac Ventricle


title: Serine Conjugated Surface 32 hours


title: N-4_PRE


title: Aspartic Acid Conjugated Surface 6 hours

description:
Cultured in DMEM with 10% FBS and penicillin (100U/ml)/streptomycin (100ug/ml). Media changed every other day. 37ºC, 5% CO2.
Cells plated on sample surfaces.


title: N-3_POST


title: N-7_PRE


title: Serine Surface 6 hours


title: N-4_POST


title: N-8_POST


title: N-9_POST


title: Glutamic Acid Surface 6 hours


title: Glutamic Acid Conjugated Surface 32 hours


title: N-22_PRE


title: N-6_POST


title: N-8_PRE


title: Aspartic Acid Conjugated Surface 32 hours


title: N-3_PRE


title: N-22_POST


title: N-7_POST


title: N-9_PRE


title: Amino terminated Surface 6 hours


title: IM-6_PRE

description:
19 paired human left ventricular apex samples were harvested at the time of implant of a left ventricular assist device (PRE) and at the time of explant (POST). The cohort included patients that were clinically classified as "ischemic" (I) showing evidence of coronary artery disease, "non-ischemic" (N) no evidence of coronary artery disease or "acute Myocardial  infarction" (IM) myocardial infarction within 10 days of the implant. Tissue was processed and hybridized to the Affymetrix HG-U133A chip.
Keywords = Heart
Keywords = Cardiac
Keywords = Assist Device
Keywords = LVAD
Keywords = GATA-4
Keywords = Sprouty1
Keywords = microarray
Keywords = heart failure


title: N-2_POST


title: IM-5_PRE


title: IM-1_POST


title: I-7_PRE


title: IM-7_PRE


title: IM-4_POST


title: IM-6_POST


title: IM-4_PRE


title: RNA1 Exp1 untreated

status: Public on Jan 04 2004

city: Aachen

first: Martin

last: Zenke

state: NRW

address: Universitatsklinikum Aachen, RWTH

department: Cell Biology

country: Germany

institute: Institute for Biomedical Engineering

description: Neurosphere cultures were established from individually isolated E 14.5 forebrains of mouse embryos that carry a bcl2 transgene. Single-cell suspensions were prepared, seeded at 1x105 cells/ml and treated for two days with 150 nM Trichostatin A (TSA), 500 nM 5-Aza-2’-deoxycytidine (AzaC) or both compounds, or left untreated. Two independent experiments were performed.

source_name: neurosphere cells


title: IM-1_PRE


title: I-8_POST


title: I-8_PRE


title: I-4_POST


title: IM-5_POST


title: RNA8 Exp2 AzaC/TSA treated


title: RNA7 Exp2 TSA treated


title: I-12_PRE


title: I-13_PRE


title: IM-3_PRE


title: I-13_POST


title: I-4_PRE


title: N-2_PRE


title: I-12_POST


title: IM-3_POST


title: RNA6 Exp2 AzaC treated


title: RNA4 Exp1 AzaC/TSA treated


title: RNA2 Exp1 AzaC treated


title: RNA3 Exp1 TSA treated


title: RNA5 Exp2 untreated


title: IM-7_POST


title: Patient E (U95A)

status: Public on Dec 14 2003

city: Kiel

first: Nancy

last: Mah

institute: Christian-Albrechts-University Kiel

description:
Pathohistological and clinical examination of Patient E (57 year-old female) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study.
Total RNA from each sample was used to prepare biotinylated target RNA, according to the manufacturer’s protocol (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). Briefly, 15 microg of total RNA was used to generate double-stranded cDNA using Superscript reagents (Life Technologies) and a T7-linked oligo(dT) primer. cRNA were synthesized using the Enzo Bioarray High Yield RNA transcript labeling kit from Affymetrix, resulting in biotinylated cRNA. Labeled cRNA were cleaned using Qiagen RNeasy kit and fragmented into 35-200 bp lengths using fragmentation buffer supplied by Affymetrix. Spike controls B2, bio-B, bio-C, bio-D and Cre-x were added to the hybridization cocktail before overnight hybridization at 45°C for 16 hrs. Arrays were stained and washed using the EukGE-WS2 protocol (a dual staining) before being scanned on an Affymetrix scanner. Absolute analysis (Affymetrix Microarray Analysis Suite 5.0) was performed in the arrays using the target intensity value of 150 (global scaling, no baseline), resulting in a 11.236 scaling factor for this sample.

source_name: sigmoid colon biopsy


title: Patient E

description:
Pathohistological and clinical examination of Patient E (57 year-old female) yielded no significant pathological findings.  This patient was considered as a normal control for the purposes of the study. A complete description of the RNA extraction is available under GSE405.

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. For the purposes of the microarray comparison study (GSM5994-5998), all filters were chosen from the same spotting production run and all filters were used for the first time.

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. The following output values from VisualGrid were used for subsequent data analysis: SIGNAL_RAW1 (the weighted mean pixel intensity within the node mask for the first duplicate), SIGNAL_RAW2 (the weighted mean pixel intensity within the node mask for the second duplicate), BKD_MEAN (the mean of the weighted means of all background spots in a 6 X 6 block).  For this dataset (GSM5994-5998), the weight of each pixel was set to one.

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).