title: 2.5 mg/kg 2CdA exposure

first: Thomas

last: Knudsen

summary:
In this time course pregnant CD-1 (outbred) dams were exposed to the 2-chloro analogue of 2'-deoxyadenosine (a metabolic toxin) on 8 d.p.c. The test dose of 2.5 mg/kg 2CdA was modeled for a 5% increased risk of microphthalmia in term fetuses. Sampling intervals were anchored to the conditional biomarker (p53 protein induction) expressed in 2CdA-treated embryos between 3.0- and 4.5h post-exposure. Since p53 protein induction has been defined as a critical event in 2CdA-induced micropthalmia, these sampling intervals represent times before (3.0h), during (4.5h), and after (6.0h) this critical event. All measurements were on RNA from the embryonic headfold.
Keywords = time series
Keywords = 2-chloro-2'-deoxyadenosine
Keywords = embryo
Keywords = p53 protein induction
Keywords = microphthalmia
Keywords: time-course

first: Kimberly

last: Nemeth

first: Amar

last: Singh

first: Jeffrey

middle: H

last: Charlap


title: Microarray-based CGH using BAC clones

status: Public on Feb 29 2004

city: Columbus

first: Nehad

last: Mohamed

state: OH

address: 420W12th Ave

department: Comperhensive Cancer Center/HCG

country: USA

institute: The Ohio State University

type: Genome variation profiling by genome tiling array

summary:
High resolution BAC array used to study changes in the copy number of chromosome 21 in Acute Meyloid Leukemia patients
Genomic DNA from patients as well as from normal donors were differentially labelled and hybridized on the array
36 RPCI-11 BAC clones covering the q-arm of chromosome21 from position15.1MB(close to the centromere)to the telomeric position 46.9 MB with an average gap between clones of 800 kb (range 346-1593 kb)In addition 23 randomly selected clones, each representing 1 of the remaining chromosomes1-20,22,X and YPositions of genes and BAC clones were determined according to the NCBI (http://www.ncbi.nlm.nih.gov/mapview) 59 BAC clones spotted in 6 replicas for a total 354 spots on GAPII corning glass slides using Affymetrix GMS417 Affymetrix Arrayer with 4-ring&pin configuration. (Affymetrix,Santa Clara, CA). Purified clones spotted in 50%DMSO DNA Isolation of these clones was performed from bacterial culture (250ml) using Qiagen midi kit (Qiagen, Valencia CA)
gDNA from AML patients and healthy controls was extracted and labelleb according to the published protocol by JR Pollack in Nature Genetics vol.23 Sep.99, also available http://cmgm.stanford.edu/pbrown/protocols/4_genomic.html
Fluorescence intensity ratios were measured in control experiments (reference versus reference hybridization) to weight DNA Amplification or deletion
Fluorescence ratio >1 indicates amplification, Fluorescence ratio <1 indicate deletion
Mean of the background corrected pixel-by-pixel fluorescence ratio between the two dyes at each spot was obtained by genePix software.
Series_platform_id: my_array13
Series_submitter weblink: www.dnaarrays.org
Keywords: other

first: Claudia

last: Baldus

first: Albert

middle: D

last: Chapelle

first: Herbert

last: Auer

first: Sandya

last: Liyanarachchi

first: Krzysztof

last: Mrozek

first: Stephan

middle: M

last: Tanner

first: Martin

last: Guimond

first: Amy

middle: S

last: Ruppert

first: Ramana

middle: V

last: Davuluri

first: Michael

middle: A

last: Caligiuri

first: Clara

last: Bloomfield


title: 2CdA-induced microphthalmia

status: Public on Mar 11 2004

city: Louisville

middle: B

state: KY

address: 501 S Preston St

department: Birth Defects Center

institute: University of Louisville

summary:
In this dose series pregnant CD-1 (outbred) dams were exposed to the 2-chloro analogue of 2'-deoxyadenosine (a metabolic toxin) on 8 d.p.c. Test doses were developed from a teratological series for 2CdA-induced microphthalmia using BMDS modeling software from the US EPA. Benchmark doses for microarray analysis were: 5.0 mg/kg (25% risk), 2.5 mg/kg (5% risk), 1.25 mg/kg (NOAEL), and 0.625 mg/kg (subNOAEL). All measurements were on RNA from the embryo proper at 3.0h post-treatment.
Keywords = dose series
Keywords = 2-chloro-2'deoxyadenosine
Keywords = embryo
Keywords = microphthalmia
Keywords: dose response

middle: R


title: Effect of LPS and LPS-binding protein treatment for 8 hours on RAW 264.4 cells

status: Public on Mar 05 2004

city: Pasadena

first: Sangdun

last: Choi

state: CA

address: 1200 E. California Blvd

department: Biology

institute: California Institute of Technology

summary:
All samples were gathered from mouse RAW 264.7 cells (macrophages). Control total RNA was extracted from untreated RAW 264.7 cells cultured for 8 hours. Test total RNA was extracted from lipopolysaccharide (100ng/ml) and lipopolysaccharide-binding protein (100pM) treated RAW 264.4 cells cultured for 8 hours. Experiment design included three control vs test arrays and three dye swap arrays.
Keywords: other

first: Yun Anna

last: Cao

first: Mi Sook

last: Chang

first: Sun Young

last: Lee

first: Meghdad

last: Rahdar

first: Robert

last: Hsueh


title: acute myeloid leukemia (AML)

status: Public on Feb 26 2004

first: Nehad

last: Mohamed

summary:
[Submitter has not provided a description of the experiment]
Keywords: other

first: Claudia

last: Baldus


title: acute myeloid leukemia (AML)

first: Herbert

last: Auer

address: 420 West 12th Ave

institute: Human Cancer Genetics


title: In vivo LPS effects on cardiac endothelial RNA-15K cDNA array

city: Dallas

first: John

middle: G

last: Maresh

state: TX

address: 5323 Harry Hines

department: Internal Medicine

institute: U.T. Southwestern Medical Center

summary:
Cardiac endothelial RNA from Tie-2 GFP mice exposed to intraperitoneal LPS vs. saline, 4 h. RNA amplified 2 rounds.  Experiment and dye reversal with cy5, cy3.
Keywords: parallel sample

platform_organism:
Homo sapiens
Mus musculus

first: Huaxia

last: Xu

last: Jiang

first: Ralph

last: Shohet


title: Effect of LPS and LPS-binding protein treatment for 16 hours on RAW 264.4 cells

summary:
All samples were gathered from mouse RAW 264.7 cells (macrophages). Control total RNA was extracted from untreated RAW 264.7 cells cultured for 16 hours. Test total RNA was extracted from lipopolysaccharide (100ng/ml) and lipopolysaccharide-binding protein (100pM) treated RAW 264.4 cells cultured for 16 hours. Experiment design included three control vs test arrays and three dye swap arrays.
Keywords: other


title: Reduced atherosclerosis in MyD88 null mice

city: Malmö

first: Harry

last: Björkbacka

address: CRC 91:12, UMAS entrance 72

department: Dept. of Clinical Sciences, Malmö

country: Sweden

institute: Lund University

summary:
This series represents a comparison of gene expression in mouse aorta from MyD88-/-, apoE-/- mice versus apoE-/- control mice fed a high fat diet for 8 weeks, causing atherosclerotic lesion development.
5 MyD88-/-, apoE-/- mice were compared to 5 apoE-/- control mice and dye swap replicated for a total of 10 replicate microarrays.
Keywords = Mus musculus
Keywords = aorta
Keywords = atherosclerosis
Keywords = MyD88
Keywords: other

last: Bjorkbacka

first: Vidya

last: Kunjathoor

first: Kathryn

middle: J

last: Moore

first: Stephanie

last: Koehn

first: Christine

last: Ordija

first: Melinda

first: Terry

last: Means

first: Kristen

last: Halmen

first: Andrew

last: Luster

first: Douglas

middle: T

last: Golenbock

first: Mason

middle: W

last: Freeman


title: Cardiac endothelial in vivo responses to LPS

summary:
Transcriptional responses of cardiac endothelial cells isolated from Tie-2 GFP mice 4 h and 24 h after intraperitoneal LPS injection.  Operon V1 long oligo set.  Hybridization using Cy3 and Cy5 labeled amplified RNA.
Keywords: parallel sample


title: Prim_Fibs

status: Public on Mar 01 2004

city: Bruce

first: Kristine

last: Hardy

state: ACT

address: University of Canberra

country: Australia

institute: University of Canberra

summary:
To determine changes in gene expression in primary fibroblasts undergoing replicative senescence, a direct comparison between early passage proliferating cells and senescent cells was performed. Frozen stocks of the primary fibroblasts (HMF3) from which the lines HMF3A and HMF3Dwt were derived (O'Hare, PNAS:98, 2001) were serially passaged twice through to senescence to yield 2 independent sets of RNA. A third set of RNA was from a different female donor (F1068). The three different sets of RNA were put on 4 arrays each. Data is also provided for F1068 fibroblasts at late passage.
Keywords = fibroblast
Keywords = senescence
Keywords = breast
Keywords: ordered

first: Louise

last: Mansfield

first: Alan

last: Mackay

first: Silvia

last: Benvenuti

first: Mike

last: O'Hare

first: Parmjit

last: Jat


title: GenomeTiling

status: Public on Mar 04 2004

city: Seattle

first: Stephen

last: Edwards

state: WA

department: Research Genetics

institute: Rosetta Inpharmatics (Merck & Co.)

summary:
Samples were purchased from Clontech as mRNA.
Hybridization material was generated through a random-priming amplification procedure (RP-AMP) using primers with a random sequence at the 3' end and a fixed motif at the 5' end.  shDNP256 (first strand synthesis): 5'- TAG ATG CTG TTG NNN NNN NNN -3'  shT7N9 (second strand synthesis): 5'- ACT ATA GGG AGA NNN NNN NNN -3'  1.5  g of mRNA was reverse-transcribed with Superscript II and the DP256 random primer for 20 minutes at 42 C (10 mM DTT, 50 mM Tris-HCl pH 8.3, 75 mM KCl, 8 mM MgCl2, 0.5 mM dNTPs, 5 U/ l Superscript II). The RNA was  degraded with the addition of 20 l volume of 0.5N sodium hydroxide and 0.25 M EDTA for 20 minutes at 65 C.  The single stranded cDNA was purified using a commercial kit (Qiagen Qiaquick).
The resulting product of single stranded cDNA was placed in its entirety in a second strand reaction. Second strand synthesis reactions utilized shT7N9 random primer and the Klenow fragment of DNA polymerase utilizing standard reaction conditions (37 C for 60 minutes, 0.2 mM DTT, 2.1 mM Tris-HCl pH 7.9, 2.1 mM MgCl2, 10.7 mM NaCl, 1.07 mM dNTPs, 0.1U/ l Klenow), followed by another Qiaquick purification.  Multiple polymerase chain reactions were run using 0.15  g of double stranded DNA and standard reaction conditions.  Amplification was achieved using 10 cycles of PCR with the DP256 and T7 primers (20 mM Tris-HCl pH 8.4, 50 mM KCl, 0.01 mM dNTPs, 1.5 mM MgCl2, 0.01 U/ l Taq Polymerase)  DP256: 5'- GTT CGA GAC CTC TAG ATG CTG TTG -3'  T7: 5'- AA TTA ATA CGA CTC ACT ATA GGG AGA -3'  followed by Qiaquick purification.
Further amplification was achieved using in vitro transcription reactions with X0.5  g dsDNA and T7 RNA Polymerase (7.5 mM DTT, 40 mM Tris-HCl pH 7.5, 14.25 mM MgCl2, 10 mM NaCl, 2 mM Spermidine, 125 U/ml RNAguard, 2.5 mM dNTPs, 15 U/ml IPPase, 25kU/ml T7 Polymerase) for 16 hours at 42 C. The cRNA was purified (RNeasy) and  reverse transcribed using Superscript and random 9-mers and amino-allyl dUTP (42 C for 20 minutes, 10 mM DTT, 50mM Tris-HCl pH 8.3, 75 mM KCl, 8 mM MgCl2, 0.5 mM dNTPs, 0.5 mM aa-dUTP, 5 U/ l Superscript II).
The cDNA for each channel was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 1M sodium bicarbonate, pH 9.0, followed by quenching with 4.0 M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were purified with a QIAquick PCR purification kit and the percentage dye incorporation and total cDNA yield was determined spectrophotometrically. Cy5 and Cy3 labelled cDNAs were combined and added to 2 mls 30% formamide with Herring Sperm DNA for hybridization to the microarray.
Further details can be found in Castle et al., Genome Biol 2003;4(10):R66, http://genomebiology.com/2003/4/10/R66 , Roberts CJ et al., Science 2000, 287:873-880, and Schadt, Edwards, et al., unpublished.
Normalization of single channel data is described in Ying, et al., "Identification of Chromosomal Regions Containing Transcribed Sequences Using Microarrays and Computational Methods" , 2003 Proceedings of the American Statistical Association, Biopharmaceutical Section [CD-ROM], Alexandria, VA: American Statistical Association, 2003.
Briefly, an invariant set of probes was selected based on their intensity ranks.  Then, a LOESS regression model was fit for Cy5 vs. Cy3 intensities for all probes in the invariant set.  Finally, for all probes on the array the Cy5 intensities were normalized to the corresponding Cy3 intensities using prediction values from the regression model.
Note that the normalized intensity values given here were used for all analyses reported in the accompanying paper.  The ratio values reported here were not used for the analysis described in the manuscript and are not calculated from the normalized intensity values given below.
Keywords: other

first: Eric

middle: E

last: Schadt

first: Debraj

last: GuhaThakurta

first: Dan

last: Holder

first: Lisa

last: Ying

first: Vladimir

last: Svetnik

first: Amy

last: Leonardson

first: Kyle

last: Hart

first: Archie

last: Russell

first: Guoya

last: Li

first: Guy

last: Cavet

first: John

last: Castle

first: Zhengyan

last: Kan

first: Ronghua

last: Chen

last: Kasarskis

first: Mihai

last: Margarint

last: Caceres

first: Jason

last: Johnson

first: Christopher

last: Armour

first: Philip

last: Garrett-Engele

first: Nicholas

middle: F

last: Tsinoremas

first: Daniel

last: Shoemaker


title: Expression profiling of plants overexpressing transcription factor WIN1

status: Public on Feb 28 2004

city: York

first: Pierre

last: Broun

department: Department of Biology

country: United Kingdom

institute: CNAP - University of York

summary:
T2 progenies of two transgenic lines overexpressing ERF transcription factor WIN1 were grown on soil in parallel under identical conditions.  mRNA was extracted from pooled leaves from multiple plants of each line for the microarray experiement.
Plants derived from two lines were used in this experiment: line 13, a "medium" overexpressor, and line 22, a strong overexpressor.  Control mRNA was obtained from pooled control plants transformed using an empty binary vector.
All plants were first selected on medium containing kanamycin before being transferred to soil.
Keywords = wax epidermis transcription factor Arabidopsis
Keywords: parallel sample

first: Patricia

last: Poindexter

first: Erin

last: Osborne

first: Cai-Zhong

last: Jiang

first: Jose Luis

last: Riechmann


title: Effect of LPS and LPS-binding protein treatment for 1 hour on RAW 264.4 cells

first: Sangdun

last: Choi

summary:
All samples were gathered from mouse RAW 264.7 cells (macrophages). Control total RNA was extracted from untreated RAW 264.7 cells cultured for 1 hour. Test total RNA was extracted from lipopolysaccharide (100ng/ml) and lipopolysaccharide-binding protein (100pM) treated RAW 264.4 cells cultured for 1 hour. Experiment design included three control vs test arrays and three dye swap arrays.
Keywords: other

first: Yun Anna

last: Cao

first: Mi Sook

last: Chang

first: Sun Young

last: Lee

first: Meghdad

last: Rahdar

first: Robert

last: Hsueh


title: Responses of HUVEC to Hypoxia and Reoxygenation

status: Public on Feb 18 2004

city: San Antonio

first: Phillip

last: Bowman

address: 3400 Rawley E. Chambers Ave

institute: Institute of Surgical Research

summary:
Human umbilical vein endothelial cells (HUVEC) were cultivated on 1%-gelatin-coated 75-cm2 culture flasks in Medium 200 supplemented with 2% fetal calf serum, penicillin (100 units/ml), streptomycin (100 units/ml) and Funizone (0.25 μg/ml) supplied by Cascade Biologics (Portland, OR).   Prior to an experiment, HUVECs were subcultivated with Trypsin/EDTA onto 1% gelatin coated glass 30-mm glass coverslips in 60 mm glass Petri dishes.
The hypoxic treatment was performed in airtight chambers from Mitsubishi Gas Chemical Co. Inc (Remel Scientific, Baton Rouge, LA) Preconditioning consisted of 1-hour hypoxia followed by various periods of reoxygenation (0, 3, 5, 12 and 24 hrs) for gene expression analysis.
Total RNA was extracted from cultured HUVECs with TRITM reagent according to the manufacture’s instructions (Molecular Research Center, Cincinnati, OH). Cy5 and Cy3 labeled cDNA probes were generated from total RNA by reverse transcription in the presence of aminoallyl-dUTP according to the method described by Xiang, et al,15.  Human universal reference RNA (Stratagene, La Jolla, CA) was Cy3-labeled and used as reference RNA and RNA isolated from HUVEC at 0, 3, 5, 12 and 24 hour following reoxygenation were Cy5-labeled. The high-resolution scans of hybridized microarrays were acquired with a GenePix scanner 4000B (Axon Instruments, Inc., Union City, CA) and tabulated with GenePix pro 5.1 software.

Keywords: time-course

first: Qidong

last: Tang

first: Baiteng

last: Zhao

first: James

last: Bynum


title: Systemic Leaf Wound Response in Tomato

status: Public on Feb 17 2004

city: Rehovot

last: Fluhr

department: Plant Sciences

country: Israel

institute: Weizmann Institute of Science

summary:
Wild type systemic leaf wound response vs. response in mutants lacking NADPH oxidase (Rboh)
Keywords = reactive oxygen species
Keywords: other

first: Moshe

last: Sagi

first: Olga

last: Davydov

first: Saltanat

last: Orazova

first: Zhazira

last: Yesbergenova

first: Ron

last: Ophir

first: Johannes

last: Stratmann


title: rlm1 mutant 4 hours exposure to congo red

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)

summary:
S. cerevisiae rlm1Δ strain was grown on YEPD. For Congo Red 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 Congo Red to a final concentration of 30 μg/ml. Cells were collected at 4 hours of growth, frozen at -80 °C and processed for RNA extraction.

Keywords: repeat sample

first: Raúl

last: García

first: Clara

last: Bermejo

first: Cecilia

last: Grau

first: Rosa

last: Pérez

first: Jose

last: Rodríguez-Peña

first: Jean

last: Francois

first: César

last: Nombela

first: Jose Manuel


title: slt2 mutant 4 hours exposure to congo red

summary:
S. cerevisiae slt2Δ strain was grown on YEPD. For Congo Red 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 Congo Red to a final concentration of 30 μg/ml. Cells were collected at 4 hours of growth, frozen at -80 °C and processed for RNA extraction.

Keywords: repeat sample


title: wild type 4 hours exposure to congo red

summary:
S. cerevisiae was grown on YEPD. For Congo Red 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 Congo Red to a final concentration of 30 μg/ml. Cells were collected at 4 hours of growth, frozen at -80 °C and processed for RNA extraction.

Keywords: repeat sample


title: wild type two hours exposure to zymolyase

summary:
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.

Keywords: repeat sample


title: wild type 0 hours exposure to congo red

summary:
S. cerevisiae was grown on YEPD. For Congo Red 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 Congo Red to a final concentration of 30 μg/ml. At this time cells were collected t=0h.

Keywords: repeat sample


title: Halobacterium NRC-1 UV repair

status: Public on Feb 14 2004

first: Michael

middle: Hiroshi

last: Johnson

address: 1441 North 34th Street

department: Computational Biology

institute: Institute for Systems Biology

summary:
This project was conducted by Nitin S. Baliga of the Halobacterium group at the Institute for Systems Biology in collaboration with Jocelyne Diruggiero of University of Maryland.  Halobacterium NRC-1 cell pellets were resuspended in an isotonic buffer at a low density, placed on ice and exposed to 200J/m2 of UV-C irradiation, returned to the growth medium and allowed to recover at 42oC in dark (D) or light (L).  Total RNA was collected from each sample at 30 minutes (D30 and L30) and 60 minutes (L60 and D60).
The control (C) was processed in a manner to identical to L60 except that it did not suffer any UV-C insult.
The reference RNA was prepared from an aliquot of the same culture just prior to UV-C irradiation.  Therefore the reference was also exposed to initial perturbations prior to UV-C irradiation such as change of growth medium to buffer and change of temperature from 42oC to ice.
Keywords = ISB
Keywords = Halobacterium NRC-1
Keywords = UV repair
Keywords = ultraviolet radiation
Keywords = stress response
Keywords: other

first: Nitin

last: Baliga

first: Sarah

last: Bjork

first: Richard

last: Bonneau

first: Min

last: Pan

first: Chika

last: Iloanusi

first: Molly

last: Kottemann

first: Leroy

last: Hood

first: Jocelyne

last: DiRuggiero

first: Bruz

last: Marzolf


title: wild type 6 hours exposure to congo red

first: Javier

last: Arroyo

summary:
S. cerevisiae was grown on YEPD. For Congo Red 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 Congo Red to a final concentration of 30 μg/ml. Cells were collected at 6 hours of growth, frozen at -80 °C and processed for RNA extraction.
Keywords: repeat sample

first: Raúl

last: García

first: Clara


title: In vitro LPS response of SVEC cells

first: John

last: Maresh

summary:
SVEC cell cultures exposed to LPS vs control. 4 and 24 h.  Operon V1.  Hybridization with amplified RNA.
Keywords: parallel sample


title: wild type 2 hours exposure to congo red

summary:
S. cerevisiae was grown on YEPD. For Congo Red experiments, yest 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 Congo Red to a final concentration of 30 μg/ml. Cells were collected at 2hours of growth, frozen at -80 °C and processed for RNA extraction.

Keywords: repeat sample

last: Bermejo

first: Cecilia

last: Grau

first: Rosa

last: Pérez

first: Jose

first: Jean


title: Temporal Response of Rat Lung to Hemorrhage

status: Public on Feb 11 2004

first: Phillip

last: Bowman

summary:
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 (H) 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: time-course

first: Jill

middle: L

last: Sondeen

first: Baiteng

last: Zhao

first: Michael

last: Dubick

first: George

last: Vaughan


title: KM109_Human oligo batch II

status: Public on Feb 12 2004

city: Leiden

first: Ellen

last: Sterrenburg

address: Wassenaarseweg 72

department: Center for Human and Clinical Genetics

country: Netherlands

institute: Leiden University Medical Center

summary:
Timecourse experiment hybridized to human oligo array part II. Healthy human primary myoblasts (KM109).
Keywords: time-course


title: Atlantic Salmon Head Kidney Study

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

summary:
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: other

platform_organism:
Oncorhynchus mykiss
Salmo salar

first: Simon

last: Jones

first: Gordon

last: Brown

first: Kristian

last: von Schalburg

first: William

last: Davidson

first: Ben

last: Koop


title: Atlantic Salmon Macrophage Study

summary:
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: other


title: B cell response to Anti-IgM and CD40 treatment

status: Public on Feb 13 2004

first: Sangdun

last: Choi

summary:
Temporal analysis (60, 180, 360 min) of B cells treated with Anti-IgM alone, CD40 alone or Anti-IgM and CD40 (all in triplicates).
Keywords: other

first: Yun Anna

last: Cao

first: Mi Sook

last: Chang

first: Sun Young

last: Lee

first: Meghdad

last: Rahdar

first: Robert

last: Hsueh


title: B cell response to CD40 and lipopolysaccharide treatment

summary:
Temporal analysis (60, 180, 360 min) of B cells treated with CD40 alone, lipopolysaccharide alone or CD40 and lipopolysaccharide (all in triplicates).
Keywords: other


title: B cell response to Anti-IgM and ELC treatment

summary:
Temporal analysis (60, 180, 360 min) of B cells treated with Anti-IgM alone, ELC alone or Anti-IgM and ELC (all in triplicates).
Keywords: other


title: BLI_Fetal

status: Public on Feb 07 2004

city: Tsukuba City, Ibaraki

first: Chandana

last: Herath

address: Ikenodai 2

department: Development Biology

country: Japan

institute: National Institute of Agrobiological Sciences

summary:
The objective of this study was to profile liver gene expression in the liver of bovine fetuses throughout pregnancy. Thus, liver total RNA was obtained from fetuses in early (days 54 & 65), mid (days 135 & 150) and late (day 245) pregnancy. These test RNAs were individually hybridized with a control RNA obtained from nonpregnant cycling adult cows. So that the expression profiles given elsewhere represent liver gene expression in the fetsues compared to that in the liver of adult nonpregnant cows.
Keywords = differential liver gene expression
Keywords = bovine fetus
Keywords = hierarchical clustering
Keywords: time-course

first: Hiroko

last: Ishiwata

first: Satoshi

last: Shiojima

first: Tadashi

last: Kadowaki

first: Susumu

last: Katsuma

first: Koichi

last: Ushizawa

first: Kei

last: Imai

first: Toru

last: Takahashi

first: Akira

last: Hirasawa

first: Seiya

first: Yoshiaki

last: Izaike

first: Gozoh

last: Tsujimoto

first: Kazuyoshi

last: Hashizume


title: BLI_Fetal_NT

summary:
The objective of this study was to compare liver gene expressions in bovine fetuses derived from somatic cell nuclear transplantion (NT) with those in the liver of bovine fetuses derived from artificial insemination (AI) and in vitro fertilized (IVF) eggs. Gene expression profiles in the liver of an NT-calf was also compared with those in the liver of AI-calf.
Keywords = nuclear transplantation
Keywords = bovine
Keywords = liver gene expression
Keywords = fetus
Keywords: parallel sample


title: Heterogous hybridization experiment

status: Public on Feb 05 2004

city: Austin

first: Hans

middle: A.

last: Hofmann

address: 1 University Station - C0930

department: Section of Integrative Biology

institute: Universit of Texas at Austin

summary:
Fish and Chips: Expression Profiling in Non-traditional Model Systems Using a Cichlid Fish cDNA Microarray

This series represents the 26 arrays that went into Renn et al 2004 (submitted January 16th), and one additional hybridization (GSM15240) that was not included in the publication analysis for statistical reasons.
Keywords: other

sample_organism:
Danio rerio
Salmo salar
Poecilia reticulata
Xiphophorus
Oreochromis niloticus
Haplochromis burtoni
Neolamprologus brichardi
Xenotilapia melanogenys

first: Susan

middle: C

last: Renn

first: Nadia

last: Aubin-Horth


title: yeast filamentous-form growth

status: Public on Feb 02 2004

first: Timothy

last: Galitski

address: 1441 N. 34th Street

summary:
Wild-type diploid cells were shifted from yeast-form growth in SHAD liquid (plentiful glucose and ammonium) to filamentous-form growth on SLAD agar (low ammonium). Samples of filamentous-form cells were collected hourly for 10 hours. Filamentous-form and yeast-form exponential-phase targets were co-hybridized.
Keywords: time-course


title: liver, 24h starved

status: Public on Feb 09 2004

city: Karlsruhe

first: Michael

middle: J.

last: Pankratz

department: Institute of Genetics

country: Germany

institute: Forschungszentrum Karlsruhe

summary:
Mice used in this study were 129/sv males, 8-15 weeks of age, which were housed individually and received water ad libitum. Normal fed animals were fed before and during the 48h experiment with standard food ad libitum. Starved animals were fed before the experiment with standard food ad libitum and were starved during the 24h experiment. Mice were killed by CO2 asphyxiation. The livers were snap-frozen in liquid nitrogen and stored at -80 degrees C. Total RNA was prepared using the Nucleospin RNA L kit (Macherey-Nagel, Düren, Germany), with an additional step in which the final eluate was used for a subsequent elution step. PolyA RNA was extracted with the Ambion (Ambion Inc, Austin, USA) Purist kit according to the manufacturer's protocols. Equal amounts of polyA RNA were pooled to generate pools of all animals. Pool1, normal = 3 animals (N1, N2, N3), pool2, normal = 3 animals (N4, N5, N6), pool3, = 6 animals (N1, N2, N3, N4, N5, N6), pool1, sugar = 3 animals (St1, St2, St3), pool2, starved = 3 animals (St4, St5, St6), pool3, starved = 6 animals (St1, St2, St3, St4, St5, St6). Labeled cDNA was synthesized from 1.5 µg polyA RNA using the Amersham (Amersham Europe, Freiburg, Germany) direct cDNA labeling kit. Removal of the unincorporated dyes and concentration of the target were done with Microcon 30 (Millipore, Bedford, MA) spin columns according to the manufacturer's instructions. The concentrated probes were hybridized to the microarray in 1x dig easy hyb buffer (Hoffman-la Roche, Basel, CH) overnight at 42 degrees C. After hybridization the microarrays are washed 5’ in 2x SSC, 0.1% SDS at 42 degrees C; 10’ in 0.1x SSC, 0.1% SDS at RT; 4 times 1’ in 0.1x SSC at RT and shortly dipped into 0.01x SSC. Drying is performed in centrifuge on a microtiterplate holder at 800 rpm for 7’. Slides are stored in dark until they are scanned. Arrays were scanned using the dual laser scanner Axon 4000B and the corresponding software Genepix 4 (Axon Inc., Union City, CA, USA). Both channels (532 nm for Cy3 and 635 nm Cy5) were scanned in parallel and stored as 16 bit tiff files. The absolute intensity values span the range from 0 to 65,535. The scans were performed with a resolution of 10 µm. From each spot with a mean diameter of 100 µm, 100 data pixels were recorded. Individual local background area around the spots were defined, which included approximately 400 pixels and excludes neighboring spots. For each channel, the raw data was calculated as the median intensity of all foreground pixels with respect to all background pixels. Each array was scanned three times (low, medium and high scan) with different signal amplification factors (voltage settings of the photo multiplier tubes), but with the same laser power. The channels for Cy3 and Cy5 were balanced in each scan for approximately the same intensity profile. In the low scan no spot was saturated, in the high scan the signal amplification for Cy5 was set to approximately 80% of maximum and the Cy3 amplification was adjusted to this. The settings used in the medium scan lie between the low and the high scan. This method for scanning has several advantages. In the low scan where no spot is in saturation, it is possible to calculate the real ratio for genes with high expression levels; however, those with a low expression level are most likely not recognized. In order not to lose these, a high scan is made; in this case, the information on the saturated spots is lost, so the two scans complement each other. The medium scan produces additional values for subsequent calculations. By scanning the arrays three times, errors which occur while recording and which might increase the error factor in the normalization are averaged. The data processing was automated in a Visual Basic/Excel (Microsoft) program, which integrates the following steps. Raw data are derived from the result files generated by scanning and picture analysis software Genepix. The spot diameter is to lie in the range from 80 to 120 µm otherwise these spots are not included in analysis. From the pixel to pixel ratios between the foreground values of both colors, the standard deviation (SD) is calculated. Those spots which show a ratio SD of higher than 3 are excluded from analysis due to inconsistency. The foreground signal of each spot is corrected by subtracting the corresponding local background. Resulting values which are lower than the background are replaced by the background value. This defines the minimum measurement threshold as the background and avoids calculations of completely wrong ratios, e.g., if the background corrected value for one channel is close to zero. Spots are marked as saturated in one channel if more than 10% of the foreground pixels are at the highest absolute value. From the unsaturated data points of all three scans, linear regression between the low and medium, and between low and high scans, are calculated for both colors. Taking these regressions, the values of saturated spots are replaced by estimated ones which reflect the real intensity. The generated data set of each scan is normalized afterwards based on the intensity-dependent methods as described by Yang et al (80). The ratios are calculated as log2 transformed values: M=log2 (Cy5/Cy3). Number describing the spot intensity A=log2sqrt (Cy5*Cy3) is calculated according to Yang et al (80). A within pin-tip group loess fit to the MA-plot was made. The loess scatter plot smoother performs robust locally fits using a tricube function to weight the values relative to the median point in the intensity interval. As interval size 20% was chosen, which corresponds to 180 data points in a pin-tip group containing 30 by 30 spots. After combining the normalized data from all blocks the data sets of all scans are additionally normalized using a global approach. The normalization was performed so that the sum of all log transformed ratios (M) is 0. All statistical normalizations are based on the assumption that (1) the majority of genes are not changed in their expression and (2) that the overall up and down regulations statistically compensate each other in sum. Taking this into account, the standard deviation in the ratios of the stable genes (80% of the most unchanged genes) could be seen as the measurement noise of the array experiments. To be able to compare the different scans and different normalized microarrays, the SD of the stable genes ratios was adjusted to a medium estimated value of 0.2, which is dependent on the used array system. This value does not influence the subsequent statistical analysis because the normal distribution of the data is not changed. For each microarray the normalized and adjusted log ratios of the three scans are averaged.
Keywords = nutrient response
Keywords = starvation
Keywords = liver
Keywords = anti-aging
Keywords = longevity
Keywords: repeat sample

first: Matthias

last: Bauer

first: Anne

last: Hamm

first: Joerg

middle: D

last: Katzenberger


title: liver, 48h sugar

summary:
Mice used in this study were 129/sv males, 8-15 weeks of age, which were housed individually and received water ad libitum. Normal fed animals were fed before and during the 48h experiment with standard food ad libitum. Sugar fed animals were fed before the experiment with standard food ad libitum and during the 48h experiment with 50% (w/v) sugar solution (40% glucose, 10% sucrose). Mice were killed by CO2 asphyxiation. The livers were snap-frozen in liquid nitrogen and stored at -80 degrees C. Total RNA was prepared using the Nucleospin RNA L kit (Macherey-Nagel, Düren, Germany), with an additional step in which the final eluate was used for a subsequent elution step. PolyA RNA was extracted with the Ambion (Ambion Inc, Austin, USA) Purist kit according to the manufacturer's protocols. Equal amounts of polyA RNA were pooled to generate pools of all animals. Pool1, normal = 3 animals (N1, N2, N3), pool2, normal = 3 animals (N4, N5, N6), pool3, = 4 animals (N7, N8, N9, N10), pool1, sugar = 3 animals (Su1, Su2, Su3), pool2, sugar = 3 animals (Su4, Su5, Su6), pool3, sugar = 4 animals (Su7, Su8, Su9, Su10). Labeled cDNA was synthesized from 1.5 µg polyA RNA using the Amersham (Amersham Europe, Freiburg, Germany) direct cDNA labeling kit. Removal of the unincorporated dyes and concentration of the target were done with Microcon 30 (Millipore, Bedford, MA) spin columns according to the manufacturer's instructions. The concentrated probes were hybridized to the microarray in 1x dig easy hyb buffer (Hoffman-la Roche, Basel, CH) overnight at 42 degrees C. After hybridization the microarrays are washed 5’ in 2x SSC, 0.1% SDS at 42 degrees C; 10’ in 0.1x SSC, 0.1% SDS at RT; 4 times 1’ in 0.1x SSC at RT and shortly dipped into 0.01x SSC. Drying is performed in centrifuge on a microtiterplate holder at 800 rpm for 7’. Slides are stored in dark until they are scanned. Arrays were scanned using the dual laser scanner Axon 4000B and the corresponding software Genepix 4 (Axon Inc., Union City, CA, USA). Both channels (532 nm for Cy3 and 635 nm Cy5) were scanned in parallel and stored as 16 bit tiff files. The absolute intensity values span the range from 0 to 65,535. The scans were performed with a resolution of 10 µm. From each spot with a mean diameter of 100 µm, 100 data pixels were recorded. Individual local background area around the spots were defined, which included approximately 400 pixels and excludes neighboring spots. For each channel, the raw data was calculated as the median intensity of all foreground pixels with respect to all background pixels. Each array was scanned three times (low, medium and high scan) with different signal amplification factors (voltage settings of the photo multiplier tubes), but with the same laser power. The channels for Cy3 and Cy5 were balanced in each scan for approximately the same intensity profile. In the low scan no spot was saturated, in the high scan the signal amplification for Cy5 was set to approximately 80% of maximum and the Cy3 amplification was adjusted to this. The settings used in the medium scan lie between the low and the high scan. This method for scanning has several advantages. In the low scan where no spot is in saturation, it is possible to calculate the real ratio for genes with high expression levels; however, those with a low expression level are most likely not recognized. In order not to lose these, a high scan is made; in this case, the information on the saturated spots is lost, so the two scans complement each other. The medium scan produces additional values for subsequent calculations. By scanning the arrays three times, errors which occur while recording and which might increase the error factor in the normalization are averaged. The data processing was automated in a Visual Basic/Excel (Microsoft) program, which integrates the following steps. Raw data are derived from the result files generated by scanning and picture analysis software Genepix. The spot diameter is to lie in the range from 80 to 120 µm otherwise these spots are not included in analysis. From the pixel to pixel ratios between the foreground values of both colors, the standard deviation (SD) is calculated. Those spots which show a ratio SD of higher than 3 are excluded from analysis due to inconsistency. The foreground signal of each spot is corrected by subtracting the corresponding local background. Resulting values which are lower than the background are replaced by the background value. This defines the minimum measurement threshold as the background and avoids calculations of completely wrong ratios, e.g., if the background corrected value for one channel is close to zero. Spots are marked as saturated in one channel if more than 10% of the foreground pixels are at the highest absolute value. From the unsaturated data points of all three scans, linear regression between the low and medium, and between low and high scans, are calculated for both colors. Taking these regressions, the values of saturated spots are replaced by estimated ones which reflect the real intensity. The generated data set of each scan is normalized afterwards based on the intensity-dependent methods as described by Yang et al (80). The ratios are calculated as log2 transformed values: M=log2 (Cy5/Cy3). Number describing the spot intensity A=log2sqrt (Cy5*Cy3) is calculated according to Yang et al (80). A within pin-tip group loess fit to the MA-plot was made. The loess scatter plot smoother performs robust locally fits using a tricube function to weight the values relative to the median point in the intensity interval. As interval size 20% was chosen, which corresponds to 180 data points in a pin-tip group containing 30 by 30 spots. After combining the normalized data from all blocks the data sets of all scans are additionally normalized using a global approach. The normalization was performed so that the sum of all log transformed ratios (M) is 0. All statistical normalizations are based on the assumption that (1) the majority of genes are not changed in their expression and (2) that the overall up and down regulations statistically compensate each other in sum. Taking this into account, the standard deviation in the ratios of the stable genes (80% of the most unchanged genes) could be seen as the measurement noise of the array experiments. To be able to compare the different scans and different normalized microarrays, the SD of the stable genes ratios was adjusted to a medium estimated value of 0.2, which is dependent on the used array system. This value does not influence the subsequent statistical analysis because the normal distribution of the data is not changed. For each microarray the normalized and adjusted log ratios of the three scans are averaged.
Keywords = nutrient response
Keywords = starvation
Keywords = liver
Keywords = anti-aging
Keywords = longevity
Keywords: repeat sample

last: Gähler


title: liver, 48h starved

summary:
Mice used in this study were 129/sv males, 8-15 weeks of age, which were housed individually and received water ad libitum. Normal fed animals were fed before and during the 48h experiment with standard food ad libitum. Starved animals were fed before the experiment with standard food ad libitum and were starved during the 48h experiment. Mice were killed by CO2 asphyxiation. The livers were snap-frozen in liquid nitrogen and stored at -80 degrees C. Total RNA was prepared using the Nucleospin RNA L kit (Macherey-Nagel, Düren, Germany), with an additional step in which the final eluate was used for a subsequent elution step. PolyA RNA was extracted with the Ambion (Ambion Inc, Austin, USA) Purist kit according to the manufacturer's protocols. Equal amounts of polyA RNA were pooled to generate pools of all animals. Pool1, normal = 3 animals (N1, N2, N3), pool2, normal = 3 animals (N4, N5, N6), pool3, = 4 animals (N7, N8, N9, N10), pool1, sugar = 3 animals (St1, St2, St3), pool2, starved = 3 animals (St4, St5, St6), pool3, starved = 4 animals (St7, St8, St9, St10). Labeled cDNA was synthesized from 1.5 µg polyA RNA using the Amersham (Amersham Europe, Freiburg, Germany) direct cDNA labeling kit. Removal of the unincorporated dyes and concentration of the target were done with Microcon 30 (Millipore, Bedford, MA) spin columns according to the manufacturer's instructions. The concentrated probes were hybridized to the microarray in 1x dig easy hyb buffer (Hoffman-la Roche, Basel, CH) overnight at 42 degrees C. After hybridization the microarrays are washed 5’ in 2x SSC, 0.1% SDS at 42 degrees C; 10’ in 0.1x SSC, 0.1% SDS at RT; 4 times 1’ in 0.1x SSC at RT and shortly dipped into 0.01x SSC. Drying is performed in centrifuge on a microtiterplate holder at 800 rpm for 7’. Slides are stored in dark until they are scanned. Arrays were scanned using the dual laser scanner Axon 4000B and the corresponding software Genepix 4 (Axon Inc., Union City, CA, USA). Both channels (532 nm for Cy3 and 635 nm Cy5) were scanned in parallel and stored as 16 bit tiff files. The absolute intensity values span the range from 0 to 65,535. The scans were performed with a resolution of 10 µm. From each spot with a mean diameter of 100 µm, 100 data pixels were recorded. Individual local background area around the spots were defined, which included approximately 400 pixels and excludes neighboring spots. For each channel, the raw data was calculated as the median intensity of all foreground pixels with respect to all background pixels. Each array was scanned three times (low, medium and high scan) with different signal amplification factors (voltage settings of the photo multiplier tubes), but with the same laser power. The channels for Cy3 and Cy5 were balanced in each scan for approximately the same intensity profile. In the low scan no spot was saturated, in the high scan the signal amplification for Cy5 was set to approximately 80% of maximum and the Cy3 amplification was adjusted to this. The settings used in the medium scan lie between the low and the high scan. This method for scanning has several advantages. In the low scan where no spot is in saturation, it is possible to calculate the real ratio for genes with high expression levels; however, those with a low expression level are most likely not recognized. In order not to lose these, a high scan is made; in this case, the information on the saturated spots is lost, so the two scans complement each other. The medium scan produces additional values for subsequent calculations. By scanning the arrays three times, errors which occur while recording and which might increase the error factor in the normalization are averaged. The data processing was automated in a Visual Basic/Excel (Microsoft) program, which integrates the following steps. Raw data are derived from the result files generated by scanning and picture analysis software Genepix. The spot diameter is to lie in the range from 80 to 120 µm otherwise these spots are not included in analysis. From the pixel to pixel ratios between the foreground values of both colors, the standard deviation (SD) is calculated. Those spots which show a ratio SD of higher than 3 are excluded from analysis due to inconsistency. The foreground signal of each spot is corrected by subtracting the corresponding local background. Resulting values which are lower than the background are replaced by the background value. This defines the minimum measurement threshold as the background and avoids calculations of completely wrong ratios, e.g., if the background corrected value for one channel is close to zero. Spots are marked as saturated in one channel if more than 10% of the foreground pixels are at the highest absolute value. From the unsaturated data points of all three scans, linear regression between the low and medium, and between low and high scans, are calculated for both colors. Taking these regressions, the values of saturated spots are replaced by estimated ones which reflect the real intensity. The generated data set of each scan is normalized afterwards based on the intensity-dependent methods as described by Yang et al (80). The ratios are calculated as log2 transformed values: M=log2 (Cy5/Cy3). Number describing the spot intensity A=log2sqrt (Cy5*Cy3) is calculated according to Yang et al (80). A within pin-tip group loess fit to the MA-plot was made. The loess scatter plot smoother performs robust locally fits using a tricube function to weight the values relative to the median point in the intensity interval. As interval size 20% was chosen, which corresponds to 180 data points in a pin-tip group containing 30 by 30 spots. After combining the normalized data from all blocks the data sets of all scans are additionally normalized using a global approach. The normalization was performed so that the sum of all log transformed ratios (M) is 0. All statistical normalizations are based on the assumption that (1) the majority of genes are not changed in their expression and (2) that the overall up and down regulations statistically compensate each other in sum. Taking this into account, the standard deviation in the ratios of the stable genes (80% of the most unchanged genes) could be seen as the measurement noise of the array experiments. To be able to compare the different scans and different normalized microarrays, the SD of the stable genes ratios was adjusted to a medium estimated value of 0.2, which is dependent on the used array system. This value does not influence the subsequent statistical analysis because the normal distribution of the data is not changed. For each microarray the normalized and adjusted log ratios of the three scans are averaged.
Keywords = nutrient response
Keywords = starvation
Keywords = liver
Keywords = anti-aging
Keywords = longevity
Keywords: repeat sample


title: rat small intestine samples

status: Public on Jan 28 2004

city: Newark

first: Xuelin

last: Cui

state: NJ

address: 185 S. Orange Ave.

department: Pharmacology and Physiology

institute: New Jersey Medical School-UMDNJ

summary:
Experiment description

1.	Experimental design

1)	Authors, laboratory, and contact: Xue-Lin Cui1, Patricia Soteropoulos2, Peter Tolias2, Ronaldo P. Ferraris1: 1Dept. of Pharmacology and Physiology, UMDNJ-New Jersey Medical School, Newark, NJ 07103-2714, 2Center for Applied Genomics, PHRI, 225 Warren Street Newark, NJ 07103-3506. Contact to Dr. Ronaldo P. Ferraris.
2)	Type of the experiment: treated vs. untreated comparison.
3)	Experimental factors: Twenty to twenty-two old SD rats were use in the experiment. The intestines of paired pups were perfused with 100 mM fructose (HF) and 100 mM glucose (HG) for 4-h or 20-min, respectively.
4)	Totally 14 experiments were done.
5)	A common reference was not used for all the hybridizations.
6)	Quality control steps ( 14)  Miller RA, Galecki A, Shmookler-Reis RJ. Interpretation, design, and analysis of gene array expression experiments. J Gerontol A Biol Sci Med Sci. 2001 Feb;56(2):B52-7.            (15)      Tseng GC, Oh MK, Rohlin L, Liao JC, Wong WH. Issues in cDNA microarray analysis: quality filtering, channel normalization, models of variations and assessment of gene effects. Nucleic Acids Res. 2001 Jun 15;29(12):2549-57.          ( 16)         Yang YH, Speed T. Design issues for cDNA microarray experiments. Nat Rev Genet. 2002 Aug;3(8):579-88.       (17)       Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation.  Nucleic Acids Res. 2002 Feb 15;30(4):e15.):
a) In the 4 h perfusion experiment, the intestines of the paired pups were perfused with 100 mM fructose (HF) and 100 mM glucose (HG) for 4-h, respectively. Totally five replicates were conducted. The RNA samples were extracted from HF- and HG-perfused intestines (n=5 pairs) then labelled with Cy 5 (HF1, HF2, HF3, HF4, and HF5) and Cy 3 (HG1, HG2, HG3, HG4, and HG5), respectively. At the same time, one dye swap experiment,  in which the samples extracted from HF- and HG-perfused intestines were labelled with Cy 3 (HF1) and Cy 5 (HG1), respectively, was done and used in the data analysis.
b) In the 20 min perfusion experiment, the intestines of  of the paired pups were perfused with 100 mM fructose (HF) and 100 mM glucose (HG) for 20-min, respectively. Totally four replicates were conducted. In two of the four-replicate experiments, the samples extracted from HF- and HG-perfused intestines were labelled with Cy 5 (HF1a and HF3a) and Cy 3 (HG1a and HG3a), respectively. In the other two of the four-replicate experiments, the dye was swapped, that is, the samples extracted from HF- and HG-perfused intestines were labelled with Cy 3 (HF2a and HF4a) and Cy 5 (HG2a and HG4a), respectively.
c) Three self-self experiments using different samples (HF1, HG1, and HF2) and one capture primer alone experiment were conducted to eliminate the false positive genes and aid to analyze the data.
7)	The goal of the experiment: Fructose induced the expression of GLUT5 mRNA in the small intestinal epithelial cells. But it is not clear about the mechanisms involved in the phenomenal. In this experiment, we hypothesize: the expression of some genes must be changed after fructose perfusion for 4-h, these genes may regulate GLUT5 gene transcription; the expression of some genes must be changed even after 20-min fructose perfusion, these genes may trigger the GLUT5 gene expression as early response gene. Our goal in this study is to find out these potencial gene.
8)	Links (URL), citations

2.	Samples used, extract preparation, labeling and hybridization

1)	Bio-source properties
a)	Sprague Dawley rats (NCBI taxonomy);
b)	The rat was anesthetized using urathene. The rat body was kept at 37 oC under light.
c)	Descriptors relevant to the particular sample: the jejunum of the 20-d old rat pups (unknown sex) was perused.
2)	Biomaterial manipulations: the jejunum was in vivo perfused with 100 mM HF or HG for 4-h or 20-min. At the end of each experiment, the jejunum was immediately cut out and snap frozen in liqued nitrogen, and then stored the tissue in -80 oC freezer.
3)	RNA isolation: A RNeasy Midi Kit (QIAGEN Inc., Valencia, CA) was used to extract total RNA from the jejunum. The brief procesure is described in the Total RNA Extraction for RT-PCR and Microarray above.
4)	Sample assignment to slides for labeling and hybridization:
S1: HF1(Cy 5) vs HG1 (Cy 3); S2: HF2 (Cy 5) vs HG2 (Cy 3); S3: HF3 (Cy 5) vs HG3 (Cy 3); S4: HF4 (Cy 5) vs HG4 (Cy 3); S5: HF5 (Cy 5) vs HG5 (Cy 3); S6: HF2 (Cy 3) vs HG2 (Cy 5); S7: HF1a (Cy 5) vs HG1a (Cy 3); S8: HF3a (Cy 5) vs HG3a (Cy 3); S9: HF2a  (Cy 3) vs HG2a (Cy 5); S10: HF4a (Cy 3) vs HG4a (Cy 5); S11: HG1 (Cy 5) vs HG1 (Cy 3); S12: HG3 (Cy 5) vs HG3 (Cy 3); S13: HF3 (Cy 5) vs HF3 (Cy 3); S14: Cy 3 vs Cy 5 capture primer.
5)	Labeling and hybridization procedures and parameters:
The 3DNA Submicro Oligo Expression Array Detection Kit (Genisphere Inc., Hatfield, PA) was employed to synthesize and label the complimentary DNA (cDNA) with fluorescent dye: Cy3 or Cy5. The reason why this indirect labeling method was chosen is based on a previous study which demonstrated that the method was more sensitive and consistent than direct labeling method or other indirect techniques like aminoallyl labeling method and MICROMAX tyramide signal amplification labeling method. (18)Yu J, Othman MI, Farjo R, et al: Molecular Vision 2002; 8:130-137). In the primary experiments, three, four, and five  g of total RNA were tested for labeling and hybridization conditions. In the official experiments, four  g of total RNA isolated from each sample were used for labeling and hybridization, because the signal to noise ratio is high at this concentration. In addition, at this concentration, all most of the low expressed genes are detectable and the highly expressed genes are not fully saturated. The procedure was conducted following the manufacturer's protocol. Briefly, the first strand cDNA was synthesized using a specific poly T Cy3 RT primer or a specific poly T Cy5 RT primer in different tubes for paired samples. Combine the Cy3 and Cy5 reactions in one tube and precipitate the sample in ethanol. After briefly drying the pellet, resuspend the pellet with 40  l of hybridization buffer. Subsequently, apply the hybridization mixture onto the array slide and hybridize overnight at 55 oC in a humidified chamber. Following the hybridization, the slides were washed by placing them into 50 ml conical tubes containing 2 x SSC, 0.2% SDS for 15 min, 2 x SSC for 10 min, 0.2 x SSC for 10 min, and 95% ethanol for 1 min all at room temperature, except for the first wash, in which the temperature was 42 oC. After drying the slides, the Cy3 and Cy5 capture (connected to dye molecues) reagents mixed in hybridization buffer was pipeted onto the slides, and then hybridize at 65 oC for 3 h following slides wash as described above.

3. Measurement data and spesifications of data processing

1)	Raw data description
Slides were scanned for Cy3 and Cy5 fluorescence using an Axon GenePix4000 microarray reader (GenePix 4000A, Axon instruments, Foster City, CA) and quantitated using GenePix software. Scan parameters: Scan power: Cy 3 : Cy 5 = 100:100; Laser power: Cy 3 : Cy 5 = 1.15:1.76; Spatial resolution: ???; Pixel size: 10 or 100 ???; Pixel number: F pixels: 50-100; B pixels: 400-500 ???; PMT voltage: 600-750 for Cy 5 channel, 650-850 for Cy 3 channel.

2)	Image analysis and quantitation
For the image analysis, the density of the spots was detected using Gene Spring (Silicon Genetics, Redwood City, CA) software (GeniPix 4.0) (19) Axon Instruments Inc. (1999) GenePix 4000A User's Guide. Axon Instruments, Union City, CA). It is commercially available. Description of the algorithm and all the parameters used: R = red channel signal = F635; G = green channel signal = B 532; M = log2(R/G); A = 1/2xlog2(RxG); User-difined parameter f = 40%. For the complete image analysis out put of each image, see the NCBI's GEO database (www.ncbi.nlm.nih.gov/geo).

3)	Normalized and summarized data-gene expression data matrix
For the normalization, the following two steps were taken.
a)	When measuring the density of the spots using Gene Spring software, the ratio of Cy 3 and Cy 5 dyes was adjusted as 1 as a globle normalization for the whole slide.
b)	For the gene expression analysis, the results (midian of the density of the pixel in one spot was used) were normalized by a LOWESS (locally weighed scatter smoother) software provided by the Center for Applied Genomics, so that the results can be compared among the experiments performed at different times. (20)Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation.  Nucleic Acids Res. 2002 Feb 15;30(4):e15.).
c)	The following rules were made to decide the genes that were significantly changed: 1) Eliminate the genes with high density (over than 2X of background) in the 3DNA capture probe experiments; 2) Eliminate the genes with density lower than 2X of background ( 21)    Lee HM, Greeley GH Jr, Englander EW. Age-associated changes in gene expression patterns in the duodenum and colon of rats. Mech Ageing Dev. 2001 Apr 15;122(4):355-71.); 3) Eliminate the genes with change over than 1.5X in dye flip-over experiment; 4) Eliminate the genes with opposite results in one experiments between dye flip-over comparisons; 5) Only the genes significantly changed over 3 in 5 comparisons in 4 h perfusion experiment and over 2 in 4 comparisons in 20 min perfusion experiment were considered as reliable and biologically important.
d)	The final results discribed in this paper were presented as Means + SE and the frequency of gene changed in the HF-perfused intestine.  About the image, raw, and normalized data, and other detailed parameters, please see the gene expression data tables derived from the experiments in the NCBI's GEO database.

The design for the dye labeling in this experiments is as the follows: 1) 5 comparisons were conducted in the 4 h-perfused HG vs HF experiment, in which in 3 comparisons, the cDNA samples synthesized from HG- and HF-perfused intestines were labeled with Cy3 and Cy5, respectively; and in 2 comparisons, the cDNA samples synthesized from HG- and HF-perfused intestines were labeled with Cy5 and Cy3, respectively. 2) 4 comparisons were conducted in the 20 min-perfused HG vs HF experiment, in which in 2 comparisons, the cDNA samples synthesized from HG- and HF-perfused intestines were labeled with Cy3 and Cy5, respectively; and in 2 comparisons, the cDNA samples synthesized from HG- and HF-perfused intestines were labeled with Cy5 and Cy3, respectively. 3) 3 dye flip-over experiments using 1 RNA sample extracted from HF-perfused intestine and 2 samples extracted from HG-perfused intestines were performed to eliminate the false positive results induced by the dye labeling technique or other unsolved problems. 4) 2 experiments only applying 3DNA fluorescent probe, which is connected to complementary capture sequence, were performed to eliminate the false positive genes that were low expressed in the tissue.  The procedures for synthesizing the first strand cDNA, labeling the cDNA, hybridizaion, and detecting the signals were performed following the manufacturer's protocols.
Keywords: other

first: Xue-Lin

first: Patricia

last: Soteropoulos

first: Peter

last: Tolias

first: Ronaldo

middle: P

last: Ferraris


title: C. elegans heat stress time course

status: Public on Jan 18 2004

city: San Francisco

first: Coleen

last: Murphy

department: Biochemistry Department

institute: University of California, San Francisco

summary:
We sought to study the effect of heat stress on gene expression in C. elegans.  We cultured CF512 animals to adulthood at 25C (at which they are sterile).  We then induced heat stress by switching the culture temperature to 30C.
Keywords: time-course


title: Transcriptional changes in mouse extensor digitorum longus following lengthening contractions

status: Public on Jan 27 2004

city: Ypsilanti

first: Stephen

middle: James

last: McGregor

state: MI

address: 318 Porter Bldg

department: HPERD

institute: Eastern Michigan University

summary:
I.  Experimental Design:
a. Type of experiment:  Time course
b. Experimental factors:  3 month old mice subjected to lengthening contractions of the extensor digitorum longus muscle (EDL).  Samples of EDL collected at 6 and 72 h and compared to non-treated control.
c.  How many hybridizations in exp: 9
d.  Common reference used for all hyb: no
e.  Quality control steps:  All arrays used from same lot.  Triplicate hybridizations performed for each time point.

II. Samples used, extracts, preparation and labeling:
Animals.  Nine male C57BL/6 mice (3-4 mo of age, 27.8 ± 3.3 g body mass, Harlan Sprague-Dawley, Indianapolis, IN).
Muscle Injury.  The extensor digitorum longus (EDL) muscle of six mice were exposed to lengthening contractions.  Mice were anesthetized with 2% avertin (0.015 ml/kg) and supplemental doses were administered if the mouse responded to a toe pinch.  Animals were placed on a plexiglass platform that was maintained at 37°C.  The distal femur of the right hindlimb was fixed between screws and the foot was taped to the platform.  The distal tendons of the EDL were exposed by incision and tied to the lever arm of a servomotor (Aurora Scientific, Richmond Hill ON, Canada) with 4-0 silk suture.  Needle electrodes were placed adjacent to the peroneal nerve to stimulate dorsiflexor contraction (Grass Instruments, West Warwick, RI).  Maximal isometric force (Po) was determined by stimulating the dorsiflexors maximally at optimum length (Lo) for force development.
To induce muscle injury, 75 lengthening contractions were performed at 0.25 Hz for 5 min.  For each lengthening contraction, the muscle was stimulated at 150 Hz and stretched 100 ms after the initiation of stimulation.  Muscle stretch involved 20 % strain relative to Lo.  Force deficit was evaluated 10 min after the 75 contractions and then the incision was closed with 7-0 silk suture.  Animals were returned to their cage to recover until the time of sacrifice.  Prior to sacrifice by cervical dislocation, animals were anesthetized with avertin and the EDL muscles were excised and flash frozen in liquid nitrogen.  Muscles were stored in liquid nitrogen until RNA isolation.
RNA Isolation.  Total RNA was isolated from EDL muscles from control mice (n=3) and mice sacrificed at 6 h (n=3) and 72 h (n=3) after lengthening contractions according to the modified protocol of  Chomzynski et al..  Frozen muscles were added to preweighed tubes containing TriReagent (Sigma-Aldrich, St. Louis, MO).  Tissue was homogenized in TriReagent using a Tissue Tearor (Fischer, Pittsburgh, PA) at 30,000 rpm for 1 minute.  Homogenates were transferred to sterile, RNAase free microcentrifuge tubes and incubated for 10 min at room temperature.  Chloroform (0.2 ml; Sigma-Aldrich, St. Louis, MO) was added and after incubation, the samples were centrifuged for 15 min at 12,000 g and 4°C.  Total RNA was precipitated from the aqueous phase by the addition of isopropanol (Sigma-Aldrich), and pelleted by centrifugation.  Pellets were washed in ethanol (75 and 95 %) and air dried before resuspension in diethylpyrocarbonate (DEPC) treated water.  RNA concentration was determined by optical density at 260 nm.

III. Hybridization procedures and parameters:
Microarray Hybridization and Analysis.  To reduce measurement error, all membranes (Atlas mouse 1.2; Clontech, Palo Alto, CA) were prepared from the same lot.  Additionally, each membrane was hybridized only once to avoid errors associated with stripping and multiple hybridizations.  In order to minimize individual biological variability, total RNA was pooled from three animals for hybridization of arrays.  Three arrays were used for the pooled sample at each time point.  First strand cDNA probe generation from total RNA and microarray hybridization were performed according to manufacturer’s instructions (Atlas cDNA Expression Arrays User Manual, Clontech).  33P labeled cDNA was generated from sample RNA using [33P]-dATP (3000 Ci/mmol; ICN, Costa Mesa, CA) and Maloney murine leukemia virus (MMLV) reverse transcriptase (Clontech).  Labeled probes were purified using nucleospin columns (Clontech).
Array membranes were prehybridized in Express Hyb containing sheared Salmon testes DNA (Gibco BRL, Rockville, MD) for 30 min at 71°C in rotating hybridization tubes.  Radiolabeled probe (3 x 106 cpm) was incubated in denaturing solution (Clontech) for 20 min at 71°C, after which Cot1 DNA/neutralizing solution was added and incubation continued for 10 min.  The probe mixture was added to the hybridization tube and incubated with rotation overnight.
Following hybridization, membranes were washed four times for 30 min in 2X saturated sodium citrate (SSC) and 1 % sodium dodecyl sulphate (SDS) at 71°C, once for 30 min in 0.1X SSC and 0.5% SDS at 71°C, and once for 5 min in 2X SSC at room temperature.  Membranes were removed and immediately wrapped in plastic and exposed to a phosphor storage screen (Molecular Dynamics, Sunnyvale, CA) for four days.

IV. Measurement data and specifications:
Data Analysis.  The membrane image was acquired using a Storm phosporimager (Molecular Dynamics) and Image Quant software.  Array images were analyzed using Atlas Image 2.0 (Clontech) software to determine raw intensity levels of expression.  Raw intensity data was imported into GeneSpring (Silicon Genetics, Redwood City, CA) expression analysis software.  Intensity levels were normalized to the median expressed intensity on each of the arrays and averaged for the three arrays at each time point.  Normalized expression levels for control arrays were established as a value of 1 and data from 6 and 72 h were expressed as fold changes relative to control.
In order to simplify interpretation of the resulting dataset, a k-means cluster analysis was employed to group genes based on similarities in patterns of expression.
To reduce the likelihood of false positives, the data was filtered using a criterion 2 fold change in expression prior to clustering.  Miller et al. (61) have calculated that using an array of 10,000 features with a coefficient of variation (CV) of 20 percent, a 2 fold criterion for altered gene expression would result in zero false positives.  The mean and median CV (15.8 and 14.3%, respectively) for all arrays in the current study fell within these guidelines for the elimination of false positives.

Keywords: time-course

last: Koh

first: Francis

middle: X

last: Pizza


title: B cell response to CD40 and IL4 treatment

city: Pasadena

first: Sangdun

last: Choi

institute: California Institute of Technology

summary:
Temporal analysis (60, 180, 360 min) of B cells treated with CD40 alone, IL4 alone or CD40 and IL4 (all in triplicates).
Keywords: other

first: Yun Anna

last: Cao

first: Mi Sook

last: Chang

first: Sun Young

last: Lee

first: Meghdad

last: Rahdar

first: Robert

last: Hsueh


title: B cell response to Anti-IgM and terbutaline treatment

summary:
Temporal analysis (60, 180, 360 min) of B cells treated with Anti-IgM alone, terbutaline alone or Anti-IgM and terbutaline (all in triplicates).
Keywords: other


title: KM109_Human oligo batch I

first: Ellen

last: Sterrenburg

summary:
Timecourse experiment of healthy human primary myoblast cells (KM109)
Keywords: time-course


title: Day 7 anti-GBM mouse

status: Public on Jan 15 2004

summary:
Control total RNA, pooled from the kidney of five normal BALB/c mice, was used as a reference against total RNA extracted from the kidney of BALB/c mice injected with anti-GBM antibody for 7 days.
Keywords: repeat sample

first: Ju-Han

last: Kim

first: Il Soo

last: Ha

first: Chang-Il

last: Hwang

first: Young-Ju

first: Ji-Hoon

first: Seung-Hee

last: Yang

first: Yon Su

first: Dong-Sup

first: Woong-Yang

last: Park


title: Neutrophil transcript abundance following 3/6 hr culture in normoxia, hypoxia and anoxia

status: Public on Jan 17 2004

city: Cambridge

first: Cristin

last: Print

institute: Cambridge University

summary:
Neutrophils are key effector cells of the innate immune response and are required to migrate and function within highly adverse micro-environmental conditions.  These inflammatory sites are characterised by low levels of oxygen.  We examined the relative transcript abundance detected in an apoptosis targeted gene array following invitro neutrophil culture (3/6 hrs)  in normoxic, hypoxic and anoxic conditions.
Keywords: repeat sample

first: Sarah

middle: R

last: Walmsley

first: Walmsley

last: Sarah

first: Print

last: Cristin


title: Day 11 anti-GBM mouse

first: Sangdun

summary:
Control total RNA, pooled from the kidney of five normal BALB/c mice, was used as a reference against total RNA extracted from the kidney of BALB/c mice injected with anti-GBM antibody for 11 days.
Keywords: repeat sample

first: Yun Anna


title: Adult aging in C. elegans

first: Coleen

last: Murphy

summary:
We wished to study normal adult aging in C. elegans.  We competitively hybridized a series of experimental samples (experimental variable: adult age) against a common reference sample.

We use the CF512 fer-15(b26) II; fem-1 (hc17) IV mutant strain, which has defective spermatids which fail to activate into spermatozoa at 25C.  Culturing this strain at 25C prevented self-fertilization and therefore eliminated contributions from embryonic transcripts.  CF512 animals develop an otherwise-normal germ line; their lifespan and aging are similar to wild-type animals.

We axenized eggs and then synchronized animals via L1 arrest. Populations were very tightly synchronized.  Animals were cultured at 25C.  Samples were extracted and hybridized as described in protocols at http://microarrays.org.
Keywords = aging
Keywords: time-course


title: fumonisin biosynthesis

status: Public on Jan 02 2004

city: West Lafayette

first: Charles

middle: P.

last: Woloshuk

state: IN

institute: Purdue University

summary:
In the study, expression profiles in wild type and a fcc1 mutant were compared to identify genes with patterns of expression similar to the FUM genes. DNA microarrays were constructed with ESTs from cDNA libraries obtained from the fcc1 mutant and wild-type strain of F. verticillioides grown on maize kernels . By microarray methodology, gene expression was measured in the fcc1 mutant and the wild type during growth on maize and in a synthetic medium at pH 3 and 8. The results of the analysis provide potential candidate genes for future studies to determine their functional roles in fumonisin biosynthesis.
Keywords: other

first: AM

last: Pirttilä

first: L

middle: M

last: McIntyre

first: G

middle: A

last: Payne

first: C


title: Bacillus anthracis Stern 34F2 sporulation

status: Public on Jan 08 2004

city: Rockville

first: David

last: Rasko

state: MD

address: 9712 Medical Center Drive

department: Microbial Genomics

institute: The Institute for Genomic Research

summary:
Timecourse from log phase growth to sporulation in Bacillus anthracis Stern 34F2
Keywords: time-course

first: Hongbin

last: Liu

first: Nicholas

middle: H

last: Bergman

first: Brendan

last: Thomason

first: Shamira

last: Shallom

first: Alyson

last: Hazen

first: Joseph

last: Crossno

first: Jacques

last: Ravel

first: Timothy

last: Read

first: Scott

middle: N

last: Peterson

first: John

last: Yates III

first: Philip

last: Hanna


title: Day 16 anti-GBM mouse

first: Sangdun

last: Choi

department: Biology

summary:
Control total RNA, pooled from the kidney of five normal BALB/c mice, was used as a reference against total RNA extracted from the kidney of BALB/c mice injected with anti-GBM antibody for 16 days.
Keywords: repeat sample

first: Ju-Han

last: Kim

first: Il Soo

last: Ha

first: Chang-Il

last: Hwang

first: Young-Ju

last: Lee

first: Ji-Hoon

first: Seung-Hee

last: Yang

first: Yon Su

first: Dong-Sup

first: Yun Anna

last: Cao

first: Woong-Yang

last: Park


title: Day 3 anti-GBM mouse

summary:
Control total RNA, pooled from the kidney of five normal BALB/c mice, was used as a reference against total RNA extracted from the kidney of BALB/c mice injected with anti-GBM antibody for 3 days.
Keywords: repeat sample


title: GH inj old fat (1-3)

city: Stockholm

first: Petra

middle: Elisabeth

last: Tollet-Egnell

address: CMM L8:01

department: Dept of Molecular Medicine

institute: Karolinska Institute

summary:
effect of hGH injections in old male rat adipose tissue
Keywords: repeat sample

first: Paolo

last: Parini

first: Nina

last: Ståhlberg

first: Ingrid

last: Lönnstedt

first: Norman

first: Mats

last: Rudling

first: Amilcar

last: Flores-Morales

first: Gunnar

last: Norstedt


title: GH inj old male rat muscle (1-3)

summary:
effect of hGH injections in old male rat skeletal muscle
Keywords: repeat sample


title: GH inj old liver (1-7)

summary:
effect of hGH injections in old male rat livers
Keywords: repeat sample


title: aging in male rat liver (1-7)

summary:
effect of increasing age in male rat liver
Keywords: repeat sample


title: effect of age in male rat fat (1-3)

summary:
effect of increasing age in male rat adipose tissue
Keywords: repeat sample


title: Drosophila melanogaster transcriptome life cycle

status: Public on Dec 31 2003

city: Heidelberg

first: Boris

last: Beckmann

address: Im Neuenheimer Feld 580

department: Funktionelle Genomanalyse

institute: Deutsches Krebsforschungszentrum

summary:
Time course analysis series in Development of the transcriptome from Drosophila melanogaster using the Heidelberg FlyArray. All stages were hybridized against embryonic stage 0-4 h as reference control.
Keywords: time-course


title: aging in male rat muscle (1-3)

summary:
effect of aging in male rat skeletal muscle
Keywords: repeat sample


title: effect of age liver

summary:
effect of aging in old male rat liver
Keywords: repeat sample


title: Cardiopulmonary bypass (CPB) vs sham-operated controls

status: Public on Dec 20 2003

city: Durham

first: Mihai

middle: Victor

last: Podgoreanu

state: NC

address: Genome Science Research Bldg.1, 595 LaSalle St, Ste#1027

department: Anesthesiology

institute: Duke University Medical Center

summary:
heart from rats exposed to cardiopulmonary bypass (CPB) vs sham-operated controls
Keywords: other

middle: V


title: Comparison of WT and YODA

status: Public on Dec 30 2003

city: Stanford

first: Chris

last: Somerville

institute: Carnegie Institution

summary:
Wild type and yda-2 plants were germinated and grown on commercial potting mix in walk-in chambers with constant illumination (~150 micromoles per square meter per second) at 22oC. The aerial parts of the plants were harvested at the rosette stage, prior to bolting. Total RNA was prepared using Trizol reagent (Invitrogen) with the additional steps recommended for polysaccharide-rich tissues. Hybridization probes for the GeneChip AtGenome1 microarrays (Affymetrix) were prepared from 20 mg total RNA. All procedures followed Affymetrix protocols. Briefly, cDNA was synthesized using Superscript reverse transcriptase (Invitrogen) and a T7-(dT)24 primer, purified by phenol/chloroform extraction using Phase Lock Gel tubes (Eppendorf), and precipitated with ethanol. Labeled antisense RNA was generated with a BioArray kit (Enzo) and purified using an RNeasy kit (Qiagen). 25 mg of the fragmented cRNAs were used for hybridization to the arrays. Affymetrix instruments were used for hybridization, washing, staining and scanning of the arrays according to the provided instructions.
Data from four biological replications representing tissue grown and harvested at different times was collected. The average signals of all eight experiments were scaled to the target value of 500.

Keywords = yoda
Keywords = yda
Keywords = kinase
Keywords = MAPKKK
Keywords: parallel sample

first: Wolfgang

last: Lukowitz

first: Adrienne

last: Roeder

first: Dana

last: Parmenter


title: CPB vs sham-controls

summary:
heart from rats exposed to cardiopulmonary bypass (CPB) vs sham-operated controls
Keywords = rat
Keywords = cardiac
Keywords = cytokine
Keywords = inflammation
Keywords: repeat sample


title: HS14

status: Public on Dec 16 2003

city: Berne 9

first: Martin

last: Flück

state: Bern

address: Bühlstrase 26

department: Department of Anatomy

country: Switzerland

institute: University of Berne

summary:
14 days suspended
Keywords: repeat sample

first: Silvia

last: Schmutz

first: Matthias

last: Wittwer

first: Hans

last: Hoppeler

first: Dominique

last: Desplanches


title: HS-R1

summary:
14 days suspended, 1 day reloaded
Keywords: repeat sample


title: CPB vs sham-operated controls (1.2)

summary:
hearts from rats exposed to cardiopulmonary bypass (CPB) vs sham-operated controls
Keywords = rat
Keywords = heart
Keywords = cytokine
Keywords = inflammation
Keywords: repeat sample


title: Chemokine and receptor expression by WT and LIGHT-expressing tumor tissues

city: Chicago

first: Yang-Xin

last: Fu

state: IL

department: Pathology

institute: U. of Chicago

summary:
A study to compare chemokine and receptor expression by LIGHT-positive vs. negative tumor tissue
Keywords: parallel sample

first: Yangxin

first: Ping

last: Yu


title: Muscle unloading and reloading

summary:
Examination of effect of mechanical loading induced by hindlimb suspension, and subsequent reloading, in soleus muscle in 14 day old female pathogen–free Wistar rats.
Keywords = skeletal muscle
Keywords = atrophy
Keywords: other

first: M


status: Public on Dec 18 2003

city: Seattle

first: Stephen

middle: W

last: Edwards

department: Research Genetics

institute: Rosetta Inpharmatics (Merck & Co.)

summary:
Rosetta (Merck) Expression Validated Gene Hybridizations
Keywords: other

first: Eric

middle: E

last: Schadt

first: Debraj

last: GuhaThakurta

first: Dan

last: Holder

first: Lisa

last: Ying

first: Vladimir

last: Svetnik

first: Amy

last: Leonardson

first: Kyle

last: Hart

first: Archie

last: Russell

first: Guoya

last: Li

first: Guy

last: Cavet

first: John

last: Castle

first: Zhengyan

last: Kan

first: Ronghua

last: Chen

first: Andrew

last: Kasarskis

last: Margarint

first: Mike

last: Caceres

first: Jason

last: Johnson

first: Christopher

last: Armour

first: Philip

last: Garrett-Engele

first: Nicholas

middle: F

last: Tsinoremas

first: Daniel

last: Shoemaker


title: Day 1 anti-GBM mouse

first: Sangdun

last: Choi

address: 1200 E. California Blvd

summary:
Control total RNA, pooled from the kidney of five normal BALB/c mice, was used as a reference against total RNA extracted from the kidney of BALB/c mice injected with anti-GBM antibody for 1 day.
Keywords: repeat sample

first: Ju-Han

last: Kim

first: Il Soo

last: Ha

first: Chang-Il

last: Hwang

first: Young-Ju

last: Lee

first: Ji-Hoon

first: Seung-Hee

last: Yang

first: Yon Su

first: Dong-Sup

first: Yun Anna

last: Cao

first: Woong-Yang

last: Park


title: Mus musculus cornea experiment

city: Bethesda

first: Barbara

last: Norman

address: MSC2730, 9000 Rockville Pike

department: National Eye Institute

institute: National Institutes of Health

summary:
PURPOSE: To provide a detailed gene expression profile of the normal postnatal mouse cornea. METHODS: Serial analysis of gene expression (SAGE) was performed on postnatal day (PN)9 and adult mouse (6 week) total corneas. The expression of selected genes was analyzed by in situ hybridization. RESULTS: A total of 64,272 PN9 and 62,206 adult tags were sequenced. Mouse corneal transcriptomes are composed of at least 19,544 and 18,509 unique mRNAs, respectively. One third of the unique tags were expressed at both stages, whereas a third was identified exclusively in PN9 or adult corneas. Three hundred thirty-four PN9 and 339 adult tags were enriched more than fivefold over other published nonocular libraries. Abundant transcripts were associated with metabolic functions, redox activities, and barrier integrity. Three members of the Ly-6/uPAR family whose functions are unknown in the cornea constitute more than 1% of the total mRNA. Aquaporin 5, epithelial membrane protein and glutathione-S-transferase (GST) omega-1, and GST alpha-4 mRNAs were preferentially expressed in distinct corneal epithelial layers, providing new markers for stratification. More than 200 tags were differentially expressed, of which 25 mediate transcription. CONCLUSIONS: In addition to providing a detailed profile of expressed genes in the PN9 and mature mouse cornea, the present SAGE data demonstrate dynamic changes in gene expression after eye opening and provide new probes for exploring corneal epithelial cell stratification, development, and function and for exploring the intricate relationship between programmed and environmentally induced gene expression in the cornea.
Keywords: other

middle: L

first: Janine

last: Davis

first: Joram

last: Piatigorsky


title: C14

first: Martin

last: Flück

summary:
14 days age control
Keywords: repeat sample

first: Silvia

last: Schmutz

first: Matthias

last: Wittwer

first: Hans

last: Hoppeler

first: Dominique

last: Desplanches


title: Alpha-naphtylisothiocyanate administration time-course

status: Public on Dec 13 2003

city: Chiba

first: Kensuke

last: Kojima

state: Chiba

department: Graduate School of Pharmaceutical Sciences

institute: Chiba University

summary:
Male Sprague-Dawley rats weighing 250-300 g were purchased from Japan SLC Co. (Shizuoka, Japan). The animals were housed under a daily controlled 12-h light and 12-h dark cycle at 23 °C with free access to rat chow (Japan SLC Co.) and water for 1 week prior to the experiments.
Rats were intraperitoneally injected with 75 mg/kg body weight of ANIT (alpha-naphtylisothiocyanate) dissolved in olive oil (7.5 mg/mL). Control animals received an injection of the same volume of olive oil.
Groups of three rats were sacrificed under ether anesthesia at 12, 18, 24 hr after ANIT or olive oil administration. At the time of sacrifice, the right lateral liver lobe was removed and flash-frozen in liquid nitrogen and stored at -80 °C. Total RNA isolated from frozen livers using TRIzol reagent (Invitrogen Co., Carlsbad, CA) was mixed to minimize variation among animals. Poly(A) RNA was purified using Oligotex-dT30 (Takara Shuzo Co., Ltd., Kyoto, Japan) in accordance with manufacturer’s instructions. Fluorescence-labeled probes were prepared by reverse transcription using a superscript II reverse transcriptase (Invitrogen Co.) and cyanine-3- and cyanine-5-dUTP (Perkin-Elmer Inc., Wellesley, MA). Poly(A) RNAs derived from rats that has administrated ANIT and from those that has administrated olive oil were labeled with cyanine-5 and cyanine-3, respectively, and vise varsa. Fluorescence-labeled probes were purified using a MinElute PCR Products Purification Kit (Quiagen GmbH, Hilden, Germany) in accordance with manufacturer’s instructions. Each purified probe was suspended in hybridization buffer containing 1.6 mg/mL poly(A) (Roche Diagnostics, Basel, Switzerland) and yeast tRNA (Roche Diagnostics), 0.67 mg/mL herring sperm, 16% 20 x SSC and 0.3% SDS and applied to a cDNA microarray containing 1,800 rat genes on a slide glass (Asahi Technoglass Co., Tokyo, Japan). Three house-keeping genes (GAPDH, HPRT and b-actin) and rat unrelated traits (Lambda DNA) were also spotted on the slide as internal positive and negative controls, respectively.
Hybridization was carried out twice to eliminate any dye bias. In one experiment, duplicate slides were hybridized with probes derived from rats which were administrated ANIT and control rats that had been labeld with cyanine-3 and cyanine-5, respectively. In a replicate experiment, other duplicate slides were hybridized with probes derived from ANIT-treated and control rats that had been labeled with cyanine-5 and cyanine-3, respectively (color swap). Then, the slides were cover-slipped and incubated in a sealed chamber (Asahi Technoglass Co.) for 16 hrs under a humidified (65 °C) condition. After being washed in low-stringency buffer (2 x SSC and 0.1% SDS), high-stringency buffer (0.2 x SSC and 0.1% SDS) and 0.2 x SSC and then rinsing with 99.5% ethanol, the slide was dried by centrifugation at low speed and used for scanning.
Fluorescence was scanned by using a ScanArray 4000 (Packard BioChip Technologies, Billerica, MA) and quantified by using QuantArray Software (Packard BioChip Technologies, Billerica, MA).
Keywords: time-course


title: Gene expression profile at 18hr after ANIT administration

summary:
Gene expression profile at 18hr after alpha-naphtylisothiocyanate administration. Hybridization was carried out four times including color swap to eliminate any dye bias.
Keywords: repeat sample


summary:
Gene expression profile at 12hr after common bile duct ligation. Hybridization was carried out four times including color swap to eliminate any dye bias.
Keywords: repeat sample


title: Gene expression profile at 12hr after ANIT administration

summary:
Gene expression profile at 12hr after alpha-naphtylisothiocyanate administration. Hybridization was carried out four times including color swap to eliminate any dye bias.
Keywords: repeat sample


summary:
Gene expression profile at 3hr after common bile duct ligation. Hybridization was carried out four times including color swap to eliminate any dye bias.
Keywords: repeat sample


title: Gene expression profile at 24hr after ANIT administration

summary:
Gene expression profile at 24hr after alpha-naphtylisothiocyanate administration. Hybridization was carried out four times including color swap.
Keywords: repeat sample


summary:
Gene expression profile at 1hr after common bile duct ligation. Hybridization was carried out four times including color swap to eliminate any dye bias.
Keywords: repeat sample


title: Mouse Inbred Strain Variation Muscle

status: Public on Dec 10 2003

city: Leiden

first: Rolf

last: Turk

department: Center for Human and Clinical Genetics

institute: Leiden University Medical Center

summary:
Variation in gene expression profiles was determined between mouse inbred strains in hindlimb muscle tissue. Strains under investigation were: C57Bl/6, C57Bl/10, CBA, DBA2, and BALB/c.
Keywords: other