title: Primary Breast Tumor 2154

description: No additional information

source_name: genomic DNA from primary breast tumor2154


title: Primary Breast Tumor 2138

status: Public on Aug 16 2007

type: genomic

city: London

first: Andrew

middle: E

last: Teschendorff

address: 72 Huntley Street

department: UCL Cancer Institute/Institute for Women's Health

country: United Kingdom

institute: University College London

description: No additional information

hyb_protocol: Hybridisation protocol available via the VUMC MACF website. Also described in van den Ijssel et al, NAR 2005 Dec 16; 33(22):e192

scan_protocol: Microarray Scanner G2505B (Agilent Technologies) default settings

data_processing: Spot analysis and QC was fully automated using BlueFuse version 3.1 (BlueGnome, Cambrdige UK); mode-normalisation.

molecule: genomic DNA

organism: Homo sapiens

source_name: genomic DNA from primary breast tumor2138

label_protocol: DNA labelling protocol available via the VUMC MACF website. Also described in van den Ijssel et al, NAR 2005 Dec 16; 33(22):e192

extract_protocol: Proteinase K/SDS (according to Maniatis)

tag: Primary breast tumour (Female)

source_name: genomic DNA from male reference DNA

tag: Male Reference DNA


title: Primary Breast Tumor 2096

description: No additional information

source_name: genomic DNA from primary breast tumor2096


title: Primary Breast Tumor 2114

description: No additional information

source_name: genomic DNA from primary breast tumor2114


title: Primary Breast Tumor 2101

description: No additional information

source_name: genomic DNA from primary breast tumor2101


title: Primary Breast Tumor 2107

description: No additional information

source_name: genomic DNA from primary breast tumor2107


title: Primary Breast Tumor 2097

description: No additional information

source_name: genomic DNA from primary breast tumor2097


title: Primary Breast Tumor 2100

description: No additional information

source_name: genomic DNA from primary breast tumor2100


title: Primary Breast Tumor 2098

description: No additional information

source_name: genomic DNA from primary breast tumor2098


title: Primary Breast Tumor 2110

description: No additional information

source_name: genomic DNA from primary breast tumor2110


title: Primary Breast Tumor 2118

description: No additional information

source_name: genomic DNA from primary breast tumor2118


title: Primary Breast Tumor 2153

description: No additional information

source_name: genomic DNA from primary breast tumor2153


title: Primary Breast Tumor 2134

description: No additional information

source_name: genomic DNA from primary breast tumor2134


title: Primary Breast Tumor 2124

description: No additional information

source_name: genomic DNA from primary breast tumor2124


title: Primary Breast Tumor 2105

description: No additional information

source_name: genomic DNA from primary breast tumor2105


title: Primary Breast Tumor 2095

description: No additional information

source_name: genomic DNA from primary breast tumor2095


title: Primary Breast Tumor 2091

description: No additional information

source_name: genomic DNA from primary breast tumor2091


title: Primary Breast Tumor 2087

description: No additional information

source_name: genomic DNA from primary breast tumor2087


title: Primary Breast Tumor 2077

description: No additional information

source_name: genomic DNA from primary breast tumor2077

source_name: genomic DNA from male reference DNA


title: Keratinocytes_cis-UCA_24hr_2

type: RNA

first: Kazuyo

last: Kaneko

address: 9th floor of Guy's Tower

department: St John's Institute of Dermatology

institute: King's College London

description: Keratinocytes in the third passage were grown to 80% confluent and fed 24 h before treatment.

hyb_protocol: 10ug of the fragmented cRNA was hybridised to the array followed by washing and staining with streptavidin-phycoerythin on an Affymetrix fluidics station 450.

scan_protocol: Fluorescent images were scanned using the GeneChip Scanner 3000 (Affymetrix).

data_processing: Data from the scanner were analysed using the Affymetrix GeneChip® Operating Software (GCOS). The average (trimmed mean) signal of each array was linearly scaled to a target signal value of 100.

label: Biotin

molecule: total RNA

source_name: Epidermal keratinocytes, cis-UCA, 24hr

label_protocol: Biotin-labelling was performed using in vitro transcription labelling kit (Affymetrix).

growth_protocol: The cells were cultured in serum-free keratinocyte medium supplemented with 50mg/ml pituitary extract, 2.5ng/ml epidermal growth factor, 100units/ml penicillin and 100mg/ml streptomycin (Invitrogen) at 37ºC in 5% CO2.

extract_protocol: Total RNA was extracted using RNA-Bee (Tel-Test Inc, Friendswood, TX).

treatment_protocol: Cis-urocanic acid was added to keratinocyte cultures at final concentration of 10ug/ml and the cells were incubated for 24hr.

tag: Gender:female, Age:24, Skin type:III


title: Primary Breast Tumor 2075

description: No additional information

source_name: genomic DNA from primary breast tumor2075


title: Primary Breast Tumor 2094

description: No additional information

source_name: genomic DNA from primary breast tumor2094


title: Keratinocytes_UVR_24hr_1

source_name: Epidermal keratinocytes, UVR, 24hr

treatment_protocol: Keratinocytes were washed with PBS twice, and then irradiated by solar simulator in PBS at a dose of 12 J/cm2. The cells were maintained on a water-cooled plate during the irradiation. The PBS was then replaced with the original conditioned medium.

tag: Gender

value: female, Age:23, Skin type:I


title: Primary Breast Tumor 2081

description: No additional information

source_name: genomic DNA from primary breast tumor2081


title: Primary Breast Tumor 2078

description: No additional information

source_name: genomic DNA from primary breast tumor2078


title: Primary Breast Tumor 2079

description: No additional information

source_name: genomic DNA from primary breast tumor2079


title: Keratinocytes_trans-UCA_24hr_2

source_name: Epidermal keratinocytes, trans-UCA, 24hr

treatment_protocol: Trans-urocanic acid was added to keratinocyte cultures at final concentration of 10ug/ml and the cells were incubated for 24hr.


title: Primary Breast Tumor 2086

description: No additional information

source_name: genomic DNA from primary breast tumor2086


title: Primary Breast Tumor 2083

description: No additional information

source_name: genomic DNA from primary breast tumor2083


title: Keratinocytes_trans-UCA_24hr_1


title: Keratinocytes_UVR_24hr_2

value: female, Age:24, Skin type:III


title: Keratinocytes_PBS_24hr_2

source_name: Epidermal keratinocytes, PBS control, 24hr

treatment_protocol: PBS was added to keratinocyte cultures at final concentration of 10ul/ml and the cells were incubated for 24hr.


title: Primary Breast Tumor 2080

description: No additional information

source_name: genomic DNA from primary breast tumor2080


title: Primary Breast Tumor 2084

description: No additional information

source_name: genomic DNA from primary breast tumor2084


title: Keratinocytes_cis-UCA_24hr_1


title: Keratinocytes_PBS_24hr_1

treatment_protocol: PBS was added to keratinocyte cultures at final concentration of 100ul/ml and the cells were incubated for 24hr.


title: oxygen-depleted (Wayne Growth) - 14 days. Rep 6

status: Public on Aug 15 2007

city: Stanford

first: SMD

last: Staff

state: CA

address: 300 Pasteur Drive

department: Stanford University, School of Medicine

country: USA

institute: Stanford Microarray Database (SMD)

description:
75077
Biological replicate 6 of 9. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 14 days.

hyb_protocol: Oligonucleotide microarrays were first prehybridized for 1 h in 5X SSC, 1% BSA and 0.1% SDS and washed with H2O and isopropanol. Following the prehybridization, 10 ul hybridization solution (labeled cDNA, 5 mg tRNA, 2X SSC, 25% formamide, 0.1% SDS) was hybridized overnight at 54 C.

scan_protocol: Microarrays were scanned using a GenePix 4000A scanner (Axon Instruments). The intensities of the two dyes at each spot were quantified using ScanAlyze (Michael Eisen, http://rana.lbl.gov/EisenSoftware.htm). Before scanning the microarrays for this project we first scanned a control microarray (day 0) to set the gain for the Cy3 and Cy5 channels, and then scanned the stationary phase and NRP experiments without changing the gain values. The microarray intensity values were not normalized after scanning.

data_processing: We eliminated large fluctuations in ratios, due to very low values in spots with near zero signal intensity in one channel, by adjusting the low value in these spots to a minimum noise value. The noise value for each channel was determined by calculating the average intensity value for the 20% lowest intensity spots, and then every value below this average noise value was raised to the noise value. Data processing value is Log (base 2) of the ratio of the mean of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm).

organism: Mycobacterium tuberculosis

source_name: Cell culture grew under Wayne Growth conditions for 0 days

label_protocol: A 70-mer oligonucleotide-based microarray (tuberculosis oligonucleotide set (Qiagen)) was used. Labeled cDNA was prepared as follows: 1.5 ug of total RNA and 4.4 mg of random oligonucleotide hexamers were incubated 2 min at 98C, cooled on ice, combined with Stratascript RTase buffer, 0.5mM dA,G,CTP, 0.02mM dTTP, 1.5 nmol Cy3- or Cy5-dUTP (Amersham), and 1.8 ml Stratascript RTase (Stratagene) (in a total volume of 25 ul). This mixture was incubated 10 min at 25 C and 90 min at 42 C. cDNA was purified by microcon-10 (Amicon) filtration.

growth_protocol: The following conditions have been routinely used for in vitro experiments in our laboratories and were used here to establish a baseline expression profile for M. tuberculosis in the stationary phase experiments. Cultures of M. tuberculosis clinical isolate 1254 were started at an optical density (OD) of 0.15. Cells were grown below an OD of 0.3 for at least 1-week prior to the start of an experiment. The cultures were grown in 30 ml 7H9 medium (supplemented with bovine serum albumin, NaCl, glucose, and glycerol) in 250-ml vented tissue culture flasks and shaking at 90 rpm. Entire flasks were sampled at the given number of days after the start of the experiment. The NRP experiments were conducted as described by Wayne and Hayes. Briefly, laboratory strain H37Rv was inoculated at a starting OD of 0.004 in 20 125mm test tubes containing 8-mm stir bars. The test tube tops were sealed with silicone and stirred at 120 rpm at 37 C. The contents of two test tubes were used for each time point assayed. The cells were pelleted by centrifugation for 4 min and frozen on dry ice.

extract_protocol: Cells were disrupted by three 30-sec pulses in a BioSpec Products bead beater. After 5 min at room temperature, samples were centrifuged at 13,000 x g for 45 sec, and the supernatants were extracted with 300 ul of chloroformyisoamyl alcohol (24:1) and precipitated with isopropyl alcohol. Samples were incubated 10 min to overnight at 4 C and centrifuged for 10 min at 4 C. The RNA pellets were washed with 1 ml of 75% ethanol, centrifuged at 13,000 x g for 5 min, and air-dried. After suspension of the RNA pellets in 90 ul of water, 10 ul of DNase I 10x buffer and 4 units of DNase I (Ambion, Austin, TX) were added and the samples were incubated for 30 min. Final purification of RNA was by RNeasy column (Qiagen, Chatsworth, CA).

treatment_protocol: Frozen cell pellets were suspended in 1 ml of Trizol reagent (GIBCOy BRL) and transferred to 2-ml screw-cap tubes containing 0.4-ml of 0.1-mm diameter zirconiaysilica beads (BioSpec Products, Bartlesville, OK).

source_name: Cell culture grew under Wayne Growth conditions for 14 days


title: oxygen-depleted (Wayne Growth) - 14 days. Rep 8

description:
75079
Biological replicate 8 of 9. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 14 days.


title: oxygen-depleted (Wayne Growth) - 14 days. Rep 9

description:
75080
Biological replicate 9 of 9. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 14 days.


title: oxygen-depleted (Wayne Growth) - 20 days. Rep 1

description:
75081
Biological replicate 1 of 2. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 20 days.

source_name: Cell culture grew under Wayne Growth conditions for 20 days


title: oxygen-depleted (Wayne Growth) - 80 days. Rep 3

description:
75087
Biological replicate 3 of 5. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 80 days.

source_name: Cell culture grew under Wayne Growth conditions for 80 days


title: NZ7603_vs_WCFS1

city: Wageningen

first: L.

middle: Mariela

last: Serrano

address: Nieuwe Kanaal 9A

country: Netherlands

institute: TI Food and Nutrition

hyb_protocol: Agilent hybridization protocol version 4.1

scan_protocol: Scanning protocol. Using the Scan Array Express microarray scanner. 1.Turn on the scanner and the computer (in this order) 2.Login: scanner 3.Double click the 'ScanArray Express' icon 4.Switch on lasers 1 and 3 at least 15 minutes prior to scanning arrays 5.Click the 'File' button This is to prevent you from accidentally turning off the laser after the 15 minutes warming-up phase 6.Insert the slide with the array-side up and the label towards the outside. On Agilent slides the array side is the side with the label that has the word "Agilent" on it 7.Press 'Scan | Prescan' 8.Put resolution at 50 m, select both labels used, and set PMT values for both channels at for instance 40% (Agilent arrays) 9.Press 'Start' 10.Press 'Palette,' 'Green' as soon as projection of the image has started (first dye), select the red colour as soon as scanning of the second layer (second dye) has started 11.Adjust PMT values to balance signals obtained for both channels. If necessary do a few low-resolution scans to obtain a optimal balance 12.Click the 'Scan' button again 13.Select frame for high resolution scan, adjust resolution (to 10 m) and scan the array for both dyes 14.Press 'File | Save' to save the files, 'Save all' saves both dye layers "Create a new folder for each array "'Save all' saves the signals from both layers in individual files 15.Switch off the lasers and the scanner.

data_processing: Data Processing 1.- Background correction 2.-Normalization lowess.

organism: Lactiplantibacillus plantarum

source_name: mutant trxB2

label_protocol:
cDNA synthesis and Labeling Protocol 1.4
Annealing Annealing Mix
x µl	 RNA preparation (=10 µg)
y µl	 nuclease-free water
1 µl	 Random Nonamers
11 µl	 TOTAL
1.mix Annealing Mix in a 200 µl PCR tube gently by pipetting up and down
2.incubate for 5’ 70°C
3.cool at room temperature for 10’ (annealing)
4.spin down the mixture to the bottom of the tube
5.place on ice Reverse transcription
Reverse Transcription Mix
4 µl	5x buffer
2 µl	0.1 M DTT
1 µl	 dNTP-mix
1 µl	 AA-dUTP
1 µl 	 TMIII (keep only briefly outside 20°C)
20 µl	 TOTAL
1.Mix by very gently by pipetting up and down.
2.Incubate for 3 hrs at 42°C (in PCR-machine)
3.Cool on ice for immediate purification or place at 20°C for storage
Degradation of mRNA
1.Add 2 µl 2,5 M NaOH
2.Mix (vortex) and spin down
3.Incubate for 15 min. at 37°C
4.Add 10 µl 2 M HEPES free acid
5.Mix (vortex) and spin down
6.Ready for purification or store at 20°C
Labelling of cDNA with cyanine dyes Purification of amino allyl-modified cDNA Followed Ge Healthcare kit "Amersham CyDye (Cy3/Cy5)Reactive Dye Protocols for Post Labelling Aminoallyl-cDNA and Aminoallyl-aRNA" code RPN5661
Labelling of amino allyl-modified cDNA with CyDye
1.Add the amino allyl modified cDNA (in 0.1 M sodium bicarbonate) directly into one aliquot of CyDye NHS Ester. Resuspend the ester completely by pipetting several times and transfer the solution to an amber 1.5 ml tube
2.Incubate at room temperature, in the dark for 60 to 90 minutes
3.Add 15 µl 4 M Hydroxylamine to each coupling reaction
4.Mix by stirring and incubate at room temperature, in the dark, for 15 minutes
5.Proceed directly to purification of CyDye-labelled cDNA following Ge Healthcare kit "Amersham CyDye (Cy3/Cy5)Reactive Dye Protocols for Post Labelling Aminoallyl-cDNA and Aminoallyl-aRNA" product code RPN5661

extract_protocol:
Extraction RNA
Collecting cells
Quenching Buffer
66.7 mM	HEPES (pH 6.5) 60% Methanol
Extraction Mixture (Prepared in a screw-cap tube)
-500 µll phenol/Chloroform
-30 µl 10% SDS
-30 µl 3 M NaAc (pH 5.2)
-500 mg glass-beads (75-150 µm)
-400 µl TE buffer

1.For each sample prepare a tube with Extraction Mixture
2.Harvest cells and quench them immediately in Quenching Buffer. Add 4 volumes of Quenching Buffer ( 40°C) to 1 volume of cell culture
3.Mix and store the sample at -40°C
4.Centrifuge cells at  -20 C at 9000 rpm for 10 minutes (Centrifuge RC5B)
5.Discard the supernatant; keep the cell pellet cooled and work quickly to prevent condensation of water. Transfer the pellet with a pre-cooled spatula to a screw-crap tube with Extraction Mixture
6.Mix the cell pellet thoroughly with Extraction Mixture by shaking by hand
7.Freeze the tube immediately in liquid nitrogen
8.Break cells in the Savant FastPrep FP120. Three times 40 seconds at speed 4.0 (cool cells between steps if necessary)
9.Centrifuge for 5 minutes at 4°C in an Eppendorf centrifuge at 10.000 rpm
10.Transfer 500 µl of the supernatant to a new tube. Add 400 µl chloroform (chloroform should be cooled at 4°C)
11.Centrifuge for 2 minutes in an Eppendorf centrifuge at full speed (4°C)
5.Continue with the "High Pure RNA Isolation Kit" from Roche (order # 1 828 665)
6.Incubate for 60 minutes with DnaseI, elute with 50 µl elution buffer
7.Store the RNA in at least 2 aliquots, one of 20 µl (for concentration and quality analysis and labeling) and one back-up of 30 µl at  80°C.

tag: L. plantarum strain NZ7603 trxB2::cam cultivated UNDER RESPIRATORY CONDITIONS (+OXYGEN +HEME +VITAMIN K2)

source_name: WT

tag: L. plantarum strain WCFS1 (wild type) cultivated UNDER RESPIRATORY CONDITIONS (+OXYGEN +HEME +VITAMIN K2)


title: oxygen-depleted (Wayne Growth) - 14 days. Rep 5

description:
75076
Biological replicate 5 of 9. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 14 days.


title: oxygen-depleted (Wayne Growth) - 80 days. Rep 2

description:
75086
Biological replicate 2 of 5. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 80 days.


title: oxygen-depleted (Wayne Growth) - 80 days. Rep 1

description:
75085
Biological replicate 1 of 5. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 80 days.


title: oxygen-depleted (Wayne Growth) - 30 days. Rep 2

description:
75084
Biological replicate 2 of 2. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 30 days.

source_name: Cell culture grew under Wayne Growth conditions for 30 days


title: NZ7603 AEROBE_vs_WCFS1 AEROBE

description: NZ7603 AEROBE_vs_WCFS1 AEROBE

hyb_protocol: Agilent hybridization  protocol version 4.1

tag: Lactobacillus plantarum NZ7603 (trxB2::cam ) aerobically cultivated (oxygen supplied).

tag: Lactobacillus plantarum WCFS1 aerobically cultivated (oxygen supplied).


title: NZ7608 ANAEROBE_vs_NZ7608 AEROBE

description: NZ7608 ANAEROBE_vs_NZ7608 AEROBE

tag: Lactobacillus plantarum NZ7608 (trxB1::cam) anaerobically cultivated.

tag: Lactobacillus plantarum NZ7608 (trxB1::cam) aerobically cultivated (oxygen supplied).


title: oxygen-depleted (Wayne Growth) - 30 days. Rep 1

description:
75083
Biological replicate 1 of 2. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 30 days.


title: WCFS1 AEROBE_vs_NZ7608 AEROBE

description: WCFS1 AEROBE_vs_NZ7608 AEROBE

tag: L. plantarum WCFS1aerobically cultivated (oxygen supplied)

tag: Lactobacillus plantarum NZ7608 (trxB1::cam ) aerobically cultivated (oxygen supplied).


title: WCFS1 ANAEROBE_vs_WCFS1 AEROBE

description: WCFS1 ANAEROBE_vs_WCFS1 AEROBE

tag: Lactobacillus plantarum WCFS1 anaerobically cultivated

tag: Lactobacillus plantarum WCFS1 aerobically cultivated (oxygen supplied)


title: WCFS1_vs_NZ7608

tag: L. plantarum strain WCFS1 (wild type) cultivated UNDERRESPIRATORY CONDITIONS (+OXYGEN +HEME +VITAMIN K2)

source_name: mutant trxB1

tag: L. plantarum strain NZ7608 trxB1::cam cultivated UNDER RESPIRATORY CONDITIONS (+OXYGEN +HEME +VITAMIN K2)


title: NZ7608_vs_NZ7602

source_name: overexpression trxB1

tag: L. plantarum strain NZ7602 -carrying plasmid pMS040, trxB1 Over expression- cultivated UNDER RESPIRATORY CONDITIONS (+OXYGEN +HEME +VITAMIN K2)


title: NZ7603 ANAEROBE_vs_NZ7603 AEROBE

description: NZ7603 ANAEROBE_vs_NZ7603 AEROBE

tag: Lactobacillus plantarum NZ7603 (trxB2::cam) anaerobically cultivated.

tag: Lactobacillus plantarum NZ7603 (trxB2::cam) aerobically cultivated.


title: oxygen-depleted (Wayne Growth) - 80 days. Rep 4

description:
75088
Biological replicate 4 of 5. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 80 days.


title: NZ7608 AEROBE_vs_NZ7603 AEROBE

description: NZ7608 AEROBE_vs_NZ7603 AEROBE


title: oxygen-depleted (Wayne Growth) - 20 days. Rep 2

description:
75082
Biological replicate 2 of 2. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 20 days.


title: NZ7602_vs_NZ7603


title: oxygen-depleted (Wayne Growth) - 14 days. Rep 4

description:
75075
Biological replicate 4 of 9. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 14 days.


title: oxygen-depleted (Wayne Growth) - 14 days. Rep 2

description:
75073
Biological replicate 2 of 9. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 14 days.


title: oxygen-depleted (Wayne Growth) - 14 days. Rep 3

description:
75074
Biological replicate 3 of 9. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 14 days.


title: oxygen-depleted (Wayne Growth) - 14 days. Rep 1

description:
75072
Biological replicate 1 of 9. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 14 days.


title: oxygen-depleted (Wayne Growth) - 12 days. Rep 3

description:
75071
Biological replicate 3 of 3. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 12 days.

source_name: Cell culture grew under Wayne Growth conditions for 12 days


title: oxygen-depleted (Wayne Growth) - 14 days. Rep 7

description:
75078
Biological replicate 7 of 9. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 14 days.


title: oxygen-depleted (Wayne Growth) - 12 days. Rep 2

description:
75070
Biological replicate 2 of 3. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 12 days.


title: oxygen-depleted (Wayne Growth) - 12 days. Rep 1

description:
75069
Biological replicate 1 of 3. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 12 days.


title: oxygen-depleted (Wayne Growth) - 4 days. Rep 6

description:
75059
Biological replicate 6 of 6. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 4 days.

source_name: Cell culture grew under Wayne Growth conditions for 4 days


title: oxygen-depleted (Wayne Growth) - 4 days. Rep 5

description:
75058
Biological replicate 5 of 6. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 4 days.


title: oxygen-depleted (Wayne Growth) - 10 days. Rep 2

description:
75067
Biological replicate 2 of 3. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 10 days.

source_name: Cell culture grew under Wayne Growth conditions for 10 days


title: oxygen-depleted (Wayne Growth) - 10 days. Rep 1

description:
75066
Biological replicate 1 of 3. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 10 days.


title: oxygen-depleted (Wayne Growth) - 10 days. Rep 3

description:
75068
Biological replicate 3 of 3. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 10 days.


title: oxygen-depleted (Wayne Growth) - 8 days. Rep 3

description:
75065
Biological replicate 3 of 3. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 8 days.

source_name: Cell culture grew under Wayne Growth conditions for 8 days


title: oxygen-depleted (Wayne Growth) - 6 days. Rep 3

description:
75062
Biological replicate 3 of 3. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 6 days.

source_name: Cell culture grew under Wayne Growth conditions for 6 days


title: oxygen-depleted (Wayne Growth) - 6 days. Rep 1

description:
75060
Biological replicate 1 of 3. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 6 days.


title: Comparative genomic indexing-A. butzleri strain RM4467-2

city: Albany

first: Craig

middle: T

last: Parker

address: 800 Buchanan St

department: PSM

institute: USDA ARS

description: Intensity ratios of DNA (channel 1) divided by DNA (channel 2) signals minus respective background signals. Total array is 16 blocks arranged in 4X4 configuration.

hyb_protocol: Labeled reference and sample DNAs were combined in 45 µl Pronto! cDNA hybridization solution (Corning, Corning, NY) and heated to 95°C for 5 min. Then, 15 µl of the hybridization mixture was put onto a microarray slide and sealed with a cover slip. The microarray slide was placed in a hybridization chamber (Corning) and incubated at 42°C for 18 h. Following hybridization, the slides were washed twice in 2× SSC, 0.1% sodium dodecyl sulfate at 42°C for 10 min, followed by twice in 1× SSC at room temperature for 10 min, and finally twice in 0.2× SSC at room temperature for 5 min. The microarray slides were dried by centrifugation at 300 × g for 10 min before scanning.

scan_protocol: Axon GenePix4000B, 10nm resolution. GenePix software. Optimize intensities manually.

data_processing: Data normalization was performed by bringing the median ratio (Cy5/Cy3) for each of the 16 blocks to 1 using the following equation: (Ti) = (Cy5i/Cy3i) - c, where T is the normalized ratio, i is the gene index, Cy5 and Cy3 are the red and green intensities and c is the 50th percentile of all Cy5/Cy3 ratios. Flagged features are omitted from the data normalization process.

organism: Aliarcobacter butzleri RM4018

source_name: Arcobacter butzleri RM4018

label_protocol: Labeling protocol described in Parker et al. J Clin Microbiol 2006, 44(11):4125-4135. PMID: 16943349

extract_protocol: Genomic DNA isolated using protocol described in Parker et al. J Clin Microbiol 2006, 44(11):4125-4135. PMID: 16943349

tag: DNA labelled with Cy5-dUTP and laser scanned at 635nm with background subtracted from feature signal

organism: Aliarcobacter butzleri

source_name: Arcobacter butzleri strain RM4467

tag: DNA labelled with Cy3-dUTP and laser scanned at 532nm with background subtracted from feature signal.


title: Comparative genomic indexing-A. butzleri strain RM1588-1

source_name: Arcobacter butzleri strain RM1588

tag: DNA labelled with Cy3-dUTP and laser scanned at 532nm with background subtracted from feature signal


title: Comparative genomic indexing-A. butzleri strain RM4018-2

tag: DNA labelled with Cy5-dUTP and laser scanned at 635nm with background subtracted from feature signal.


title: oxygen-depleted (Wayne Growth) - 8 days. Rep 1

description:
75063
Biological replicate 1 of 3. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 8 days.

source_name: Cell culture grew under Wayne Growth conditions for 0 days


title: oxygen-depleted (Wayne Growth) - 4 days. Rep 3

description:
75056
Biological replicate 3 of 6. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 4 days.


title: Comparative genomic indexing-A. butzleri strain RM4596-2

source_name: Arcobacter butzleri strain RM4596


title: Comparative genomic indexing-A. butzleri strain RM1588-2


title: oxygen-depleted (Wayne Growth) - 4 days. Rep 2

description:
75055
Biological replicate 2 of 6. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 4 days.


title: oxygen-depleted (Wayne Growth) - 8 days. Rep 2

description:
75064
Biological replicate 2 of 3. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 8 days.


title: Comparative genomic indexing-A. butzleri strain RM4467-1


title: Comparative genomic indexing-A. butzleri strain RM4843-2

source_name: Arcobacter butzleri strain RM4843


title: Comparative genomic indexing-A. butzleri strain RM4018-1


title: oxygen-depleted (Wayne Growth) - 6 days. Rep 2

description:
75061
Biological replicate 2 of 3. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 6 days.


title: oxygen-depleted (Wayne Growth) - 4 days. Rep 1

description:
75054
Biological replicate 1 of 6. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 4 days.


title: Comparative genomic indexing-A. butzleri strain RM4462-1

source_name: Arcobacter butzleri strain RM4462


title: Comparative genomic indexing-A. butzleri strain RM5516-2

source_name: Arcobacter butzleri strain RM5516


title: Comparative genomic indexing-A. butzleri strain RM4596-1


title: Comparative genomic indexing-A. butzleri strain RM4850-1

source_name: Arcobacter butzleri strain RM4850


title: Growth curve 60 days (Control). Rep 3

description:
75108
Biological replicate 3 of 3. Growth curve 60 days (Control).

source_name: Cell culture grew for 0 days

source_name: Cell culture grew for 60 days


title: Comparative genomic indexing-A. butzleri strain RM4462-2


title: Growth curve 60 days (Control). Rep 1

description:
75106
Biological replicate 1 of 3. Growth curve 60 days (Control).


title: Growth curve 60 days (Control). Rep 2

description:
75107
Biological replicate 2 of 3. Growth curve 60 days (Control).


title: Comparative genomic indexing-A. butzleri strain RM5516-1


title: Growth curve 24 days (Control). Rep 4

description:
75105
Biological replicate 4 of 4. Growth curve 24 days (Control).

source_name: Cell culture grew for 24 days


title: Growth curve 24 days (Control). Rep 2

description:
75103
Biological replicate 2 of 4. Growth curve 24 days (Control).


title: Growth curve 24 days (Control). Rep 3

description:
75104
Biological replicate 3 of 4. Growth curve 24 days (Control).


title: Comparative genomic indexing-A. butzleri strain RM5538-2

source_name: Arcobacter butzleri strain RM5538


title: Growth curve 8 days (Control). Rep 3

description:
75098
Biological replicate 3 of 3. Growth curve 8 days (Control).

source_name: Cell culture grew for 8 days


title: Growth curve 8 days (Control). Rep 2

description:
75097
Biological replicate 2 of 3. Growth curve 8 days (Control).


title: Comparative genomic indexing-A. butzleri strain RM4128-2

source_name: Arcobacter butzleri strain RM4128


title: Growth curve 14 days (Control). Rep 1

description:
75099
Biological replicate 1 of 3. Growth curve 14 days (Control).

source_name: Cell culture grew for 14 days


title: Comparative genomic indexing-A. butzleri strain RM554-1

source_name: Arcobacter butzleri strain RM5544


title: Growth curve 14 days (Control). Rep 2

description:
75100
Biological replicate 2 of 3. Growth curve 14 days (Control).


title: Comparative genomic indexing-A. butzleri strain RM5544-2

description: Intensity ratios of DNA (channel 1) divided by DNA (channel 2) signals minus respective background signals. Total array is 16 blocks arranged in 4X4 configuration.

source_name: Arcobacter butzleri RM4018


title: Comparative genomic indexing-A. butzleri strain RM1591-2

source_name: Arcobacter butzleri strain RM1591


title: Comparative genomic indexing-A. butzleri strain RM1591-1


title: Comparative genomic indexing-A. butzleri strain RM4128-1


title: Growth curve 14 days (Control). Rep 3

description:
75101
Biological replicate 3 of 3. Growth curve 14 days (Control).


title: Growth curve 24 days (Control). Rep 1

description:
75102
Biological replicate 1 of 4. Growth curve 24 days (Control).


title: Comparative genomic indexing-A. butzleri strain RM5541-2

source_name: Arcobacter butzleri strain RM5541


title: H/W rat liver: 96hTCDD vs 3hTCDD

city: Toronto

first: Ivy

last: Moffat

state: Ontario

address: 1 King's College Circle, Medical Science Building Rm.4302

department: Pharmacology

country: Canada

institute: University of Toronto

description: Liver was harvested from rats treated by gavage with a single dose of 100 µg/kg TCDD or corn oil vehicle for 3 or 96 h

hyb_protocol: Hybridization was performed using Exiqon’s MiRCury LNA array hybridization kit (version 1.2) (Exiqon, Vedbaek, Denmark) according to the manufacturer's instructions.  Slides were hybridized at 60ºC for 17 h.

scan_protocol: Arrays were scanned at 10-micron resolution on a GenePix 4000B Array Scanner (Axon instruments, Inc., Union City, CA) using auto-PMT calculation settings. Each element was located and analyzed with the GenePix Pro 6.0.1.26 software package.

data_processing: Using LimmaGUI (v1.8.1; limma, v2.9.1; (Wettenhall and Smyth, 2004)), the array data were loaded into R statistical package (v2.4.0), investigated for spatial and distributional homogeneity, background subtracted (minimum method), normalized within-arrays (robust spline) and normalized between arrays (scale).

organism: Rattus norvegicus

source_name: Total RNA  from H/W rat liver, TCDD-treated for 96 h

label_protocol: Labeling of 2.5 µg of total RNA were performed using Exiqon’s MiRCury LNA array labeling kit (version 1.2)  (Exiqon, Vedbaek, Denmark) according to the manufacturer's instructions.

growth_protocol: TCDD-sensitive L-E rats (which express wildtype AHR) and TCDD-resistant H/W rats (with a deletion in the AHR transactivation domain, (Pohjanvirta et al., 1998)) were from breeding colonies of the National Public Health Institute, Division of Environmental Health, Kuopio, Finland.

extract_protocol: Total RNA was isolated from rat livers using the mirVana miRNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer’s instructions except flashPAGE fractionation (Ambion) was not performed due to high technical variability.   Total RNA yield was quantified by UV spectrophotometry and RNA integrity was verified using an Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA).

treatment_protocol: Rats were treated by gavage with a single dose of 100 µg/kg TCDD or corn oil vehicle for 3, 19, or 96 h.

tag: Strain

value: H/W

tag: Sex

value: male

tag: Treatment

tag: Genotype

value: AhrH/W/H/W

tag: Age

value: 10-12 weeks

tag: Tissue

value: liver

source_name: Total RNA  from H/W rat liver, TCDD-treated for 3 h


title: H/W rat liver: 96hTCDD vs control

source_name: Total RNA  from H/W rat liver, corn oil vehicle-treated for 19 h

value: corn oil


title: H/W rat liver: 3hTCDD vs 96hTCDD


title: H/W rat liver: control vs 3hTCDD


title: H/W rat liver: 3hTCDD vs control


title: Comparative genomic indexing-A. butzleri strain RM5538-1

label_protocol: Labeling protocol described in Parker et al. J Clin Microbiol 2006, 44(11):4125-4135.

extract_protocol: Genomic DNA isolated using protocol described in Parker et al. J Clin Microbiol 2006, 44(11):4125-4135.


title: Growth curve 6 days (Control). Rep 1

description:
75093
Biological replicate 1 of 3. Growth curve 6 days (Control).

source_name: Cell culture grew for 6 days


title: Growth curve 6 days (Control). Rep 3

description:
75095
Biological replicate 3 of 3. Growth curve 6 days (Control).


title: L-E rat liver: 3hTCDD vs control replicate3

description: Liver was harvested from rats treated by gavage with a single dose of 100 µg/kg TCDD or corn oil vehicle for 3h

source_name: Total RNA  from L-E rat liver, TCDD-treated for 3 h

value: L-E

value: AhrWt/Wt

source_name: Total RNA  from L-E rat liver, corn oil vehicle-treated for 19 h


title: L-E rat liver: control vs 3hTCDD replicate4


title: L-E rat liver: control vs 3hTCDD replicate2


title: Growth curve 0 days (Control). Rep 3

description:
75092
Biological replicate 3 of 3. Growth curve 0 days (Control).

source_name: Cell culture grew for 0 days


title: Growth curve 8 days (Control). Rep 1

description:
75096
Biological replicate 1 of 3. Growth curve 8 days (Control).


title: Growth curve 6 days (Control). Rep 2

description:
75094
Biological replicate 2 of 3. Growth curve 6 days (Control).


title: liver L-E control vs H/W control

description: Liver was harvested from rats treated by gavage with a single dose of corn oil vehicle for 19h


title: Growth curve 0 days (Control). Rep 1

description:
75090
Biological replicate 1 of 3. Growth curve 0 days (Control).


title: Growth curve 0 days (Control). Rep 2

description:
75091
Biological replicate 2 of 3. Growth curve 0 days (Control).


title: L-E rat liver: 3hTCDD vs control replicate1


title: H/W  rat liver: control vs 19hTCDD replicate4

description: Liver was harvested from rats treated by gavage with a single dose of 100 µg/kg TCDD or corn oil vehicle for 19h

source_name: Total RNA  from H/W rat liver, TCDD-treated for 19 h


title: H/W rat liver: 19hTCDD vs control replicate3


title: Comparative genomic indexing-A. butzleri strain RM5541-1


title: H/W rat liver: control vs 19hTCDD replicate2


title: liver H/W control vs L-E control


title: H/W rat liver: 19hTCDD vs control replicate1


title: L-E rat liver: 19hTCDD vs control replicate1

source_name: Total RNA  from L-E rat liver, TCDD-treated for 19 h


title: L-E rat liver: control vs 19hTCDD replicate2


title: L-E rat liver: feed restricted vs control

description: Liver was harvested from rats treated by gavage with a single dose of 100 µg/kg TCDD or corn oil vehicle or whose feed was restricted for 96 h

source_name: Total RNA  from L-E rat liver, feed restricted for 96 h

growth_protocol: TCDD-sensitive L-E rats were from breeding colonies of the National Public Health Institute, Division of Environmental Health, Kuopio, Finland.

treatment_protocol: L-E rats were treated by gavage with a single dose of 100 µg/kg TCDD,  corn oil vehicle, or their feed was restricted for 96 h.

value: 96h feed restricted

source_name: Total RNA  from L-E rat liver, corn oil vehicle treated for 96 h

value: 96h corn oil


title: L-E rat liver: 19hTCDD vs control replicate3


title: L-E rat liver: control vs 19hTCDD replicate4


title: L-E rat liver: control vs feed restricted


title: TCDD-treated Ahr+/+ mouse (WTT) liver  vs TCDD-treated Ahr-/- mouse (KOT) liver

description: Given a single dose of 1000 µg/kg TCDD or corn oil vehicle by gavage.  Liver was harvested 19 h after treatment.

organism: Mus musculus

source_name: Total RNA  from TCDD-treateted Ahr+/+ mouse (WTT) liver

extract_protocol: Total RNA was isolated from mouse liver using TRIzol reagent (Invitrogen, La Jolla, CA) according to the manufacturer’s instructions.  Total RNA yield was quantified by UV spectrophotometry and RNA integrity was verified using an Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA).

treatment_protocol: Liver tissues were from mice in which we previously mapped AHR-dependent and dioxin-dependent gene batteries by transcriptomic analysis (Tijet et al., 2006).  Briefly, male Ahr-null (Ahr-/-) mice in a C57BL/6J background (10 weeks old) and  wildtype (Ahr+/+) C57BL/6J mice (15 weeks old) were given a single dose of 1000 µg/kg TCDD or corn oil vehicle by gavage.  Liver was harvested 19 h after treatment.  We tested 3 TCDD-treated and 3 control mice in the Ahr-/- groups and 4 TCDD-treated and 4 control mice in the Ahr+/+ groups.

value: TCDD

value: Ahr+/+

value: 15 weeks

source_name: Total RNA  fromTCDD-treated Ahr-null (Ahr-/-) mouse (KOT) liver

value: Ahr-/-

value: 10 weeks


title: Ahr+/+ mouse (WT) liver: TCDD-treated vs.Control

source_name: Total RNA  from corn oil vehicle control  treateted Ahr+/+  mouse (WTC) liver


title: L-E rat liver: TCDD treated vs contol

source_name: Total RNA  from L-E rat liver, TCDD treated for 96 h


title: L-E rat liver: TCDD treated vs feed restricted


title: Day 3 + LIF embryoid bodies, Cy5

city: Cardiff

first: Fiona

middle: Catherine

last: Mansergh

state: Wales

address: Museum Avenue

department: Biosciences

institute: Cardiff University

description: Arrays were repeated twice with fluor switching, in order to counteract any issues of dye bias that may have arisen from direct labelling. 2 biological repeats were carried out (2 sets of day 0 RNA and day 1-4 +/- LIF differentiations). Spots are printed in duplicate on the slides, giving a total of 4 repeats. Scanned images were stored and filtered, then analysed using the program ImaGene™ 5.5 (BioDiscovery). Analysis and other methods were carried out similarly to another published experiment, please see below and Mansergh et al. 2004 PMID: 15303089 for more details.

hyb_protocol: Array slides were incubated in prehybe for 1 hour at 42oC (50% formamide, 5XSSC, 0.1% SDS, 1% BSA). Targets were dried down via vacuum centrifugation then resuspended in 50l hybe solution (49.9% de-ionised formamide, 49.9% 20xSSC, 0.2% SDS) with added 1l Cot1 DNA and 1l poly A oligo as blocking agents, heated to 95oC for 5 minutes and then added to the face of one slide. The printed face of the second slide of the pair was then placed face to face with the first, using the same probe. Slide pairs were then placed on a level plastic cover above some 1xSSC moistened tissue in a slide box. The slide box was sealed with Nescofilm (Karlan Research Products Corporation, Santa Rosa, CA, USA), placed floating in a waterbath and  hybridized for 24-48 hours at 42oC.  Following hybridization, slides were washed once in Wash solution 1 (1X SSC, 2% SDS, filtered autoclaved ddH2O) for 20 minutes, then twice in Wash solution 2 (0.1X SSC, 0.2% SDS, filtered autoclaved distilled deionised H2O (ddH2O) for 20 minutes each. Slides were dipped in nuclease free filtered water, then spray dried, finally, the backs of the slides were cleaned with filtered autoclaved ddH2O, then wiped with 100% EtOH, then wiped dry and scanned.

scan_protocol: Scans were carried out at 10 micrometers, using the averaging setting (GeneTac LSIV scanner, (Genomic Solutions, Cambridgeshire, UK)). It is possible to carry out quick draft scans using this scanner, gain and black settings, which affect image intensity and background were varied slightly in order to optimize the signal/noise ratio for each channel and each slide before proper scans were initiated.

data_processing:
Output files from ImaGene were saved in MS Excel spreadsheet format. MS Excel was used for all further data manipulation. We normalised each channel via total array methods, via calculation of the mean intensity value. Normalised intensities were then analysed using two different methods. Firstly, we transferred normalised intensity values for each experimental repetition into a separate Excel file, ensuring a standard order of samples. These data were then formatted for Significance Analysis of Microarrays (SAM; http://www-stat.stanford.edu/~tibs/SAM). SAM analysis was carried out and genes that showed a fold change of 2 or above and that appeared in a SAM analysis as statistically significant above a delta value of 0.5 (which denotes an error rate of 5%) were selected for further appraisal. We supplemented SAM analysis with a second analysis method as follows: The average intensities for the EMPTY spots were calculated, and this value + 2SD was used as a background cutoff figure. The data column was then normalised by division by the mean, resulting in values >1 being above average intensity and vice versa.  The mean background + 2 standard deviations of the background were divided by the normalisation factor calculated above. Fold changes were calculated for control and experimental data, furthermore, datapoints that were below background + 2sd in BOTH channels were rejected.
Meaningful data (above background + 2SD values in at least 1 channel, fold change of > 2) were retained, combined with similar data from other replicates and sorted via accession number. Genes that appeared above background + 2SD values with a fold change of > 2, in all 4 replicates were retained for further analysis. Finally, accession numbers obtained from this method and from SAM were compared. Genes appearing as differentially regulated using BOTH methods were deemed significant by us. In other words, significant ESTs are changed in expression by at least two fold, are above background + 2SD in at least one channel and have a delta value of 0.5 using SAM.  These ESTs were subjected to bioinformatic analysis and were ALL assessed via RT PCR in order to confirm array results.

molecule: cytoplasmic RNA

source_name: Day 3 + LIF embryoid bodies

label_protocol: Different RNA samples were used for labelling array repetitions. 10ug of RNA was labelled with either Cy3 or Cy5 dyes using the CyScribe labelling system (GE Healthcare, Chalfont St.Giles, Bucks, UK), according to the manufacturer’s protocol. 1ul of labelled cDNA was combined with 2ul 50% glycerol, run on a ‘John gel’ (a microscope slide sized, 1.5% agarose gel) and scanned using a GeneTac LS IV scanner (Genomic Solutions, Huntingdon, Cambs. UK) in order to assess successful incorporation of label. Control and experimental samples were then combined and prepared for hybridization.

growth_protocol: ES cells were maintained in their undifferentiated state in the medium MEM-Alpha (GibcoTM, Invitrogen Ltd, Paisley, Renfrewshire, UK) supplemented with 10-4M -Mercaptoethanol (Merck KGaA, 64293 Darmstadt, Germany), 2mM glutamine and 10-3 U/ml murine LIF (ESGROTM, Invitrogen, Ltd, Paisley, Renfrewshire, UK), 10% FBS and 10% NBS (selected batches, PAA Laboratories GmbH, Linz, A-4020 Austria). The cells were maintained at 37oC in a humidified atmosphere with 5% CO2 on 0.1% gelatin (Stem Cell Technologies) coated tissue culture grade plastic ware (NUNCTM, Fisher Scientific, Loughborough, Leics, UK).

extract_protocol: Day 0 ES cells were washed twice with DPBS-A, treated with trypsin, resuspended in 1ml medium, spun down, then washed with PBS again prior to RNA extraction. Day1 EBs were flooded with PBS from their hanging drops, transferred to a centrifuge tube, spun down and then used for RNA extraction. Days 2-4 EBs were spun down in the 20mls medium they were floating in, washed with PBS, then used for RNA extraction. The QIAgen RNeasyTM mini or midi Kits (Qiagen Ltd., Crawley, Sussex, UK) were used according to the manufacturer’s protocol for RNA extraction. These RNA samples were quantified and checked for degradation via OD 260/280 spectrophotometry (Camspec, Cambridge, UK) and gel electrophoresis using dissociating conditions (NorthernMax™ buffers; Ambion, Huntingdon, UK), used according to the manufacturer’s protocol.

treatment_protocol: See growth protocol. These ES cell lines were maintained normally, see growth protocol. They were then grown in hanging drops (160 per plate), in cell concentrations of 1000 cells per 10ul drop. After 1 day of differentiation, EBs were either flooded with 20ml medium and grown floating freely in the dish, or for Day1 samples, flooded with PBS, spun down and used with the RNEasy Mini or Midi kits for RNA extraction, according to the manufacturer’s protocol. Day 2-4 samples were spun down in medium washed in PBS and then used with the RNEasy Mini or Midi kits for RNA extraction, according to the manufacturer’s protocol. Minus LIF EBs were grown in ES medium (described in growth protocol) without the addition of LIF/ESGRO (Invitrogen). Plus LIF EBs were grown in ES medium as described below.

tag: Embryoid bodies derived from IMT-11 ES cells, day 3 of differentiation, plus LIF

source_name: Pooled control

tag: Pooled control, pool of IMT-11 embryoid body RNA isolated from all different stages and +/- LIF exposures. Targets were prepared from 10ug of this sample mix


title: Day 3 - LIF embryoid bodies, Cy3

source_name: Day 3 - LIF embryoid bodies

tag: Embryoid bodies derived from IMT-11 ES cells, day 3 of differentiation, minus LIF


title: L-E rat liver: feed restricted vs TCDD treated


title: L-E rat liver: control vs TCDD treated


title: Day 4 - LIF embryoid bodies, Cy3

source_name: Day 4 - LIF embryoid bodies

tag: Embryoid bodies derived from IMT-11 ES cells, day 4 of differentiation, minus LIF


title: Day 2 - LIF embryoid bodies, Cy3

source_name: Day 2 - LIF embryoid bodies

tag: Embryoid bodies derived from IMT-11 ES cells, day 2 of differentiation, minus LIF


title: Day 2 + LIF embryoid bodies, Cy3

data_processing:
Output files from ImaGene were saved in MS Excel spreadsheet format. MS Excel was used for all further data manipulation. We normalised each channel via total array methods, via calculation of the mean intensity value. Normalised intensities were then analysed using two different methods. Firstly, we transferred normalised intensity values for each experimental repetition into a separate Excel file, ensuring a standard order of samples. These data were then formatted for Significance Analysis of Microarrays (SAM; http://www-stat.stanford.edu/~tibs/SAM). SAM analysis was carried out and genes that showed a fold change of 2 or above and that appeared in a SAM analysis as statistically significant above a delta value of 0.5 (which denotes an error rate of 5%) were selected for further appraisal. We supplemented SAM analysis with a second analysis method as follows: The average intiensities for the EMPTY spots were calculated, and this value + 2SD was used as a background cutoff figure. The data column was then normalised by division by the mean, resulting in values >1 being above average intensity and vice versa.  The mean background + 2 standard deviations of the background were divided by the normalisation factor calculated above. Fold changes were calculated for control and experimental data, furthermore, datapoints that were below background + 2sd in BOTH channels were rejected.
Meaningful data (above background + 2SD values in at least 1 channel, fold change of > 2) were retained, combined with similar data from other replicates and sorted via accession number. Genes that appeared above background + 2SD values with a fold change of > 2, in all 4 replicates were retained for further analysis. Finally, accession numbers obtained from this method and from SAM were compared. Genes appearing as differentially regulated using BOTH methods were deemed significant by us. In other words, significant ESTs are changed in expression by at least two fold, are above background + 2SD in at least one channel and have a delta value of 0.5 using SAM.  These ESTs were subjected to bioinformatic analysis and were ALL assessed via RT PCR in order to confirm array results.

source_name: Day 2 + LIF embryoid bodies

extract_protocol: Day 0 ES cells were treated with trypsin, washed with DPBS-A resuspended in 1ml medium, spun down and washed twice with DPBS-A. The QIAgen RNeasyTM mini or midi Kits (Qiagen Ltd., Crawley, Sussex, UK) were used according to the manufacturer’s protocol for RNA extraction. These RNA samples were quantified and checked for degradation via OD 260/280 spectrophotometry (Camspec, Cambridge, UK) and gel electrophoresis using dissociating conditions (NorthernMax™ buffers; Ambion, Huntingdon, UK), used according to the manufacturer's protocol

treatment_protocol: ES cell lines were maintained normally, see growth protocol. They were then grown in hanging drops (160 per plate), in cell concentrations of 1000 cells per 10ul drop. After 1 day of differentiation, EBs were either flooded with 20ml medium and grown floating freely in the dish, or for Day1 samples, flooded with PBS, spun down and used with the RNEasy Mini or Midi kits for RNA extraction, according to the manufacturer’s protocol. Day 2-4 samples were spun down in medium washed in PBS and then used with the RNEasy Mini or Midi kits for RNA extraction, according to the manufacturer’s protocol. Minus LIF EBs were grown in ES medium (described in growth protocol) without the addition of LIF/ESGRO (Invitrogen). Plus LIF EBs were grown in ES medium as described below.

tag: Embryoid bodies derived from IMT-11 ES cells, day 2 of differentiation, plus LIF


title: Day 4 + LIF embryoid bodies, Cy5

source_name: Day 4 + LIF embryoid bodies

tag: Embryoid bodies derived from IMT-11 ES cells, day 4 of differentiation, plus LIF


title: Day 4 + LIF embryoid bodies, Cy3


title: Day 3 + LIF embryoid bodies, Cy3


title: Day 4 - LIF embryoid bodies, Cy5


title: Control Ahr-/- mouse (KOC) liver vs. Control Ahr+/+ mouse (WTC) liver

source_name: Total RNA  from corn oil vehicle control  Ahr-null (Ahr-/-) mouse (KOC) liver


title: Day 1 -LIF differentiated embryoid bodies, Cy5

source_name: Day 1 - LIF differentiated EBs

tag: Embryoid bodies derived from IMT-11 ES cells, day 1 of differentiation, minus LIF


title: H/W rat liver: control vs 96hTCDD

source_name: Total RNA  from H/W rat liver, corn oil vehicle-treated for 19 h

source_name: Total RNA  from H/W rat liver, TCDD-treated for 96 h


title: Day 3 - LIF embryoid bodies, Cy5


title: Day 2 - LIF embryoid bodies, Cy5


title: Day 2 + LIF embryoid bodies, Cy5


title: Day 1-LIF embryoid bodies (EBs), Cy3

source_name: Day 1-LIF embryoid bodies (EBs)


title: Day 1 + LIF embryoid bodies (EBs), cy5

source_name: Day 1 + LIF differentiated EBs, derived from IMT11 ES cells

tag: Embryoid bodies derived from IMT-11 ES cells, day 1 of differentiation, grown in the presence of LIF


title: oxygen-depleted (Wayne Growth) - 4 days. Rep 4

description:
75057
Biological replicate 4 of 6. Experiment grew under respiration-limited environment of the oxygen-depleted (Wayne Growth) for 4 days.

source_name: Cell culture grew under Wayne Growth conditions for 0 days

source_name: Cell culture grew under Wayne Growth conditions for 4 days


title: Ahr+/+ mouse (WT) liver: Control vs. TCDD-treated

source_name: Total RNA  from corn oil vehicle control  treateted Ahr+/+ mouse (WTC) liver

source_name: Total RNA  from TCDD-treateted Ahr+/+ mouse (WTT) liver


title: Ahr-/- mouse (KO) Liver: TCDD-treated vs control

source_name: Total RNA  fromTCDD-treated Ahr-null (Ahr-/-) mouse (KOT) liver

source_name: Total RNA  from corn oil vehicle control treated Ahr-null (Ahr-/-) mouse (KOC) liver


title: Control Ahr+/+ mouse (WTC) liver  vs Control Ahr-/- mouse (KOC) liver

source_name: Total RNA  from corn oil vehicle control  treateted Ahr+/+  mouse (WTC) liver


title: Ahr-/- mouse (KO) Liver: Control vs.TCDD-treated

source_name: Total RNA  from corn oil vehicle control treated for 19 hr Ahr-null (Ahr-/-) mouse (KOC) liver


title: TCDD-treated Ahr-/- mouse (KOT) liver  vs.TCDD-treated Ahr+/+ mouse (WTT) liver


title: 60min insulin-2

city: LOS ANGELES

first: CHIEH-FANG

last: CHENG

address: 1303 N MISSION RD

department: DERMATOLOGY

institute: USC

description: The experiment were repeated.

hyb_protocol: The samples were hybridized by following Eukaryotic Target Hybridization protocol from Affymetrix.

scan_protocol: The samples were scanned by following Eukaryotic Arrays: Washing, Staining, and Scanning protocol from Affymetrix by using GeneChip 7g 3000 scanner. GCOS software (GeneChip Operating System) were used.

data_processing: dCHIP

label: biotin

source_name: 60min, insulin

label_protocol: The samples were labeled by following 1 cycle cDNA synthesis protocol from Affymetrix.

growth_protocol: Human primary keratinocytes were cultured in Epilife supplemented with human keratinocyte growth supplements (HKGS), according to the manufacturer’s instruction. Media were changed every other day.

extract_protocol: Total RNA was extracted from HKs by using RNeasy mini kit (Qiagen) following the manufacturer’s User Manual.

treatment_protocol: Human primary keratinocytes were seeded on collagen I pre-coated tissue culture plates (150 mm) around 45% confluence (~ 2.5 X 106 cells) in complete medium and incubated overnight. Next day, the cells (~ 4.5 X 106 cells) were deprived of any supplemental GFs and incubated in serum-free medium for 16 hours to rest the cells at G0/G1 phase. The cells were then washed with pre-warmed serum-free medium and incubated in fresh serum-free medium containing 20 ng/ml TGFbeta for 20 min at 37oC to block expression of growth signal-related genes. Insulin (5microgram/ml) was added to plate (in the continued presence of TGFbeta) for 60 min. At the end of each stimulation time points, the cells were washed with ice-cold PBS to holt the stimulation and subjected to RNA isolation.

tag: Human primary keratinocyte from foreskin.

tag: HKs at passages 4 to 5 were used in all experiments throughout this study.


title: Day0 undifferentiated IMT11 ES cells

source_name: Day0 undifferentiated IMT11 ES cells

treatment_protocol: Day 0 undifferentiated ES cells, not treated, maintained as in Growth protocol

tag: Day0 undifferentiated IMT11 ES cells, plated on gelatinized plates under non-differentiating conditions

source_name: Pooled control


title: Day 1+LIF differentiated embryoid bodies (EBs), cy3

source_name: Day 1+LIF differentiated embryoid bodies (EBs), derived from IMT11 ES cells

tag: Embryoid bodies derived from IMT-11 ES cells, day 1 of differentiation, plus LIF


title: 120min control-2

description: The experiment were repeated.

source_name: control, 120min

treatment_protocol: Human primary keratinocytes were seeded on collagen I pre-coated tissue culture plates (150 mm) around 45% confluence (~ 2.5 X 106 cells) in complete medium and incubated overnight. Next day, the cells (~ 4.5 X 106 cells) were deprived of any supplemental GFs and incubated in serum-free medium for 16 hours to rest the cells at G0/G1 phase. The cells were then washed with pre-warmed serum-free medium and incubated in fresh serum-free medium containing 20 ng/ml TGFbeta for 20 min at 37oC to block expression of growth signal-related genes. PBS was added to plate (in the continued presence of TGFbeta) for 120 min. At the end of each stimulation time points, the cells were washed with ice-cold PBS to holt the stimulation and subjected to RNA isolation.


title: 120min TGFalpha-2

description: The experiment were repeated.

source_name: 120min, TGFalpha

treatment_protocol: Human primary keratinocytes were seeded on collagen I pre-coated tissue culture plates (150 mm) around 45% confluence (~ 2.5 X 106 cells) in complete medium and incubated overnight. Next day, the cells (~ 4.5 X 106 cells) were deprived of any supplemental GFs and incubated in serum-free medium for 16 hours to rest the cells at G0/G1 phase. The cells were then washed with pre-warmed serum-free medium and incubated in fresh serum-free medium containing 20 ng/ml TGFbeta for 20 min at 37oC to block expression of growth signal-related genes. TGFalpha was added to plate (in the continued presence of TGFbeta) for 120 min. At the end of each stimulation time points, the cells were washed with ice-cold PBS to holt the stimulation and subjected to RNA isolation.


title: 30min TGFalpha-2

description: The experiment were repeated.

source_name: 30min, TGFalpha

treatment_protocol: Human primary keratinocytes were seeded on collagen I pre-coated tissue culture plates (150 mm) around 45% confluence (~ 2.5 X 106 cells) in complete medium and incubated overnight. Next day, the cells (~ 4.5 X 106 cells) were deprived of any supplemental GFs and incubated in serum-free medium for 16 hours to rest the cells at G0/G1 phase. The cells were then washed with pre-warmed serum-free medium and incubated in fresh serum-free medium containing 20 ng/ml TGFbeta for 20 min at 37oC to block expression of growth signal-related genes. TGFalpha was added to plate (in the continued presence of TGFbeta) for 30 min. At the end of each stimulation time points, the cells were washed with ice-cold PBS to holt the stimulation and subjected to RNA isolation.


title: 60min control-2

description: The experiment were repeated.

source_name: control, 60min

treatment_protocol:
Human primary keratinocytes were seeded on collagen I pre-coated tissue culture plates (150 mm) around 45% confluence (~ 2.5 X 106 cells) in complete medium and incubated overnight. Next day, the cells (~ 4.5 X 106 cells) were deprived of any supplemental GFs and incubated in serum-free medium for 16 hours to rest the cells at G0/G1 phase. The cells were then washed with pre-warmed serum-free medium and incubated in fresh serum-free medium containing 20 ng/ml TGFbeta for 20 min at 37oC to block expression of growth signal-related genes.
PBS was added to plate (in the continued presence of TGFbeta) for 60 min. At the end of each stimulation time points, the cells were washed with ice-cold PBS to holt the stimulation and subjected to RNA isolation.


title: 30min TGFalpha

description: The experiment were repeated.

source_name: TGFalpha, 30min


title: Undifferentiated IMT11 ES cells, cy5

source_name: Undifferentiated IMT11 ES cells

tag: IMT11 ES cells, maintained without feeders under non-differentiating conditions


title: 60min control

description: The experiment were repeated.

treatment_protocol: Human primary keratinocytes were seeded on collagen I pre-coated tissue culture plates (150 mm) around 45% confluence (~ 2.5 X 106 cells) in complete medium and incubated overnight. Next day, the cells (~ 4.5 X 106 cells) were deprived of any supplemental GFs and incubated in serum-free medium for 16 hours to rest the cells at G0/G1 phase. The cells were then washed with pre-warmed serum-free medium and incubated in fresh serum-free medium containing 20 ng/ml TGFbeta for 20 min at 37oC to block expression of growth signal-related genes.  PBS was added to plate (in the continued presence of TGFbeta) for 60 min. At the end of each stimulation time points, the cells were washed with ice-cold PBS to holt the stimulation and subjected to RNA isolation.


title: 120min insulin

description: The experiment were repeated.

source_name: 120min, insulin

treatment_protocol: Human primary keratinocytes were seeded on collagen I pre-coated tissue culture plates (150 mm) around 45% confluence (~ 2.5 X 106 cells) in complete medium and incubated overnight. Next day, the cells (~ 4.5 X 106 cells) were deprived of any supplemental GFs and incubated in serum-free medium for 16 hours to rest the cells at G0/G1 phase. The cells were then washed with pre-warmed serum-free medium and incubated in fresh serum-free medium containing 20 ng/ml TGFbeta for 20 min at 37oC to block expression of growth signal-related genes. Insulin (5microgram/ml) was added to plate (in the continued presence of TGFbeta) for 120 min. At the end of each stimulation time points, the cells were washed with ice-cold PBS to holt the stimulation and subjected to RNA isolation.


title: 60min TGFalpha

description: The experiment were repeated.

source_name: TGFalpha, 60min

treatment_protocol: Human primary keratinocytes were seeded on collagen I pre-coated tissue culture plates (150 mm) around 45% confluence (~ 2.5 X 106 cells) in complete medium and incubated overnight. Next day, the cells (~ 4.5 X 106 cells) were deprived of any supplemental GFs and incubated in serum-free medium for 16 hours to rest the cells at G0/G1 phase. The cells were then washed with pre-warmed serum-free medium and incubated in fresh serum-free medium containing 20 ng/ml TGFbeta for 20 min at 37oC to block expression of growth signal-related genes.  TGFalpha (20ng/ml) was added to plate (in the continued presence of TGFbeta) for 60 min. At the end of each stimulation time points, the cells were washed with ice-cold PBS to holt the stimulation and subjected to RNA isolation.


title: 30min insulin-2

description: The experiment were repeated.

source_name: 30min, insulin

treatment_protocol: Human primary keratinocytes were seeded on collagen I pre-coated tissue culture plates (150 mm) around 45% confluence (~ 2.5 X 106 cells) in complete medium and incubated overnight. Next day, the cells (~ 4.5 X 106 cells) were deprived of any supplemental GFs and incubated in serum-free medium for 16 hours to rest the cells at G0/G1 phase. The cells were then washed with pre-warmed serum-free medium and incubated in fresh serum-free medium containing 20 ng/ml TGFbeta for 20 min at 37oC to block expression of growth signal-related genes. Insulin (5microgram/ml) was added to plate (in the continued presence of TGFbeta) for 30 min. At the end of each stimulation time points, the cells were washed with ice-cold PBS to holt the stimulation and subjected to RNA isolation.


title: 60min insulin

description: The experiment were repeated.

source_name: insulin, 60min

treatment_protocol: Human primary keratinocytes were seeded on collagen I pre-coated tissue culture plates (150 mm) around 45% confluence (~ 2.5 X 106 cells) in complete medium and incubated overnight. Next day, the cells (~ 4.5 X 106 cells) were deprived of any supplemental GFs and incubated in serum-free medium for 16 hours to rest the cells at G0/G1 phase. The cells were then washed with pre-warmed serum-free medium and incubated in fresh serum-free medium containing 20 ng/ml TGFbeta for 20 min at 37oC to block expression of growth signal-related genes.  insulin (5 microgram/ml) was added to plate (in the continued presence of TGFbeta) for 60 min. At the end of each stimulation time points, the cells were washed with ice-cold PBS to holt the stimulation and subjected to RNA isolation.


title: 60min TGFalpha-2

description: The experiment were repeated.

source_name: 60min, TGFalpha

treatment_protocol: Human primary keratinocytes were seeded on collagen I pre-coated tissue culture plates (150 mm) around 45% confluence (~ 2.5 X 106 cells) in complete medium and incubated overnight. Next day, the cells (~ 4.5 X 106 cells) were deprived of any supplemental GFs and incubated in serum-free medium for 16 hours to rest the cells at G0/G1 phase. The cells were then washed with pre-warmed serum-free medium and incubated in fresh serum-free medium containing 20 ng/ml TGFbeta for 20 min at 37oC to block expression of growth signal-related genes. TGFalpha was added to plate (in the continued presence of TGFbeta) for 60 min. At the end of each stimulation time points, the cells were washed with ice-cold PBS to holt the stimulation and subjected to RNA isolation.


title: 120min insulin-2

description: The experiment were repeated.


title: 30min control

description: The experiments were repeated.

scan_protocol: The samples were scanned by following Eukaryotic Arrays: Washing, Staining, and Scanning protocol from Affymetrix by using GeneChip Fluidics Station 450 and GeneChip 7g 3000 scanner. GCOS software (GeneChip Operating System) were used.

source_name: control, 30min

treatment_protocol: Human primary keratinocytes were seeded on collagen I pre-coated tissue culture plates (150 mm) around 45% confluence (~ 2.5 X 106 cells) in complete medium and incubated overnight. Next day, the cells (~ 4.5 X 106 cells) were deprived of any supplemental GFs and incubated in serum-free medium for 16 hours to rest the cells at G0/G1 phase. The cells were then washed with pre-warmed serum-free medium and incubated in fresh serum-free medium containing 20 ng/ml TGFbeta for 20 min at 37oC to block expression of growth signal-related genes. PBS was added to plate (in the continued presence of TGFbeta) for 30 min. At the end of each stimulation time points, the cells were washed with ice-cold PBS to holt the stimulation and subjected to RNA isolation.


title: 30min insulin

description: The experiment were repeated.

source_name: insulin, 30min

treatment_protocol: Human primary keratinocytes were seeded on collagen I pre-coated tissue culture plates (150 mm) around 45% confluence (~ 2.5 X 106 cells) in complete medium and incubated overnight. Next day, the cells (~ 4.5 X 106 cells) were deprived of any supplemental GFs and incubated in serum-free medium for 16 hours to rest the cells at G0/G1 phase. The cells were then washed with pre-warmed serum-free medium and incubated in fresh serum-free medium containing 20 ng/ml TGFbeta for 20 min at 37oC to block expression of growth signal-related genes.  insulin (5 microgram/ml) was added to plate (in the continued presence of TGFbeta) for 30 min. At the end of each stimulation time points, the cells were washed with ice-cold PBS to holt the stimulation and subjected to RNA isolation.


title: 120min TGFalpha

description: The experiment were repeated.

treatment_protocol:
Human primary keratinocytes were seeded on collagen I pre-coated tissue culture plates (150 mm) around 45% confluence (~ 2.5 X 106 cells) in complete medium and incubated overnight. Next day, the cells (~ 4.5 X 106 cells) were deprived of any supplemental GFs and incubated in serum-free medium for 16 hours to rest the cells at G0/G1 phase. The cells were then washed with pre-warmed serum-free medium and incubated in fresh serum-free medium containing 20 ng/ml TGFbeta for 20 min at 37oC to block expression of growth signal-related genes.
TGFalpha (20ng/ml) was added to plate (in the continued presence of TGFbeta) for 120 min. At the end of each stimulation time points, the cells were washed with ice-cold PBS to holt the stimulation and subjected to RNA isolation.


title: 120min control

description: The experiment were repeated.

treatment_protocol:
Human primary keratinocytes were seeded on collagen I pre-coated tissue culture plates (150 mm) around 45% confluence (~ 2.5 X 106 cells) in complete medium and incubated overnight. Next day, the cells (~ 4.5 X 106 cells) were deprived of any supplemental GFs and incubated in serum-free medium for 16 hours to rest the cells at G0/G1 phase. The cells were then washed with pre-warmed serum-free medium and incubated in fresh serum-free medium containing 20 ng/ml TGFbeta for 20 min at 37oC to block expression of growth signal-related genes.
PBS was added to plate (in the continued presence of TGFbeta) for 120 min. At the end of each stimulation time points, the cells were washed with ice-cold PBS to holt the stimulation and subjected to RNA isolation.


title: 30min control-2

description: The experiment were repeated.

source_name: control, 30in

treatment_protocol:
Human primary keratinocytes were seeded on collagen I pre-coated tissue culture plates (150 mm) around 45% confluence (~ 2.5 X 106 cells) in complete medium and incubated overnight. Next day, the cells (~ 4.5 X 106 cells) were deprived of any supplemental GFs and incubated in serum-free medium for 16 hours to rest the cells at G0/G1 phase. The cells were then washed with pre-warmed serum-free medium and incubated in fresh serum-free medium containing 20 ng/ml TGFbeta for 20 min at 37oC to block expression of growth signal-related genes.
PBS was added to plate (in the continued presence of TGFbeta) for 30 min. At the end of each stimulation time points, the cells were washed with ice-cold PBS to holt the stimulation and subjected to RNA isolation.


title: Activated vs Non-activated A. caninum L3 (Population IV)

city: Herston

first: Alex

middle: C

last: Loukas

state: Queensland

address: 300 Herston Rd

department: Division of Infectious Diseases and Immunology

country: Australia

institute: the Queensland Institute of Medical Research

description:
If the signal intensity in one channel was below that channel's background intensity, the signal was automatically set to background if the signal for the other channel was above that channel's background. Such an instance demonstrates an "on" versus "off" mRNA expression pattern with respect to serum stimulation.
All slides were scanned at 100% and 50% gain. If a feature was saturated in either channel (signal intensity in excess of 65254) at 100%, then the data were replaced for each channel with that from the 50% scan.

hyb_protocol: Hybridisations and washes were conducted as per Agilent’s Two-colour Gene Expression Hybridisation protocol version 5.0.1.  Briefly, using a probe volume of 250 μl of hybridisation solution was applied and the microarrays were hybridised for 17 hours at 65°C, 10 rpm overnight. The sSlides were then washed for 1 minute briefly in Gene Expression Wash Buffers 1 (RT), 1 minute  in Wash Bufferand 2 (37°C), 1 minute in Acetonitrile (RT) and 30s in Stabilisation and Drying Solution (RT) (Agilent) at 22°C and 37°C, respectively. Lastly,  slides were air-dried and scanned using a DNA Microarray Scanner (Agilent).

scan_protocol: Scanning and feature extraction were performed with an Agilent Technologies Scanner G2505B using Feature Extraction software version 9.1.3.1 (extraction protocol GE2-v4_91; Agilent).

data_processing: During extraction, signal intensities were Linear and Lowess-normalized, dye-corrected, and adjusted for local background (Feature extraction software version 9.1.3.1). Log ratios were calculated as Log2(ActL3_SIG_MEAN / NAL3_SIG_MEAN).

organism: Ancylostoma caninum

source_name: Activated

label_protocol: To generate cRNA, 200 ng of total RNA extracted from each activated and non-activated L3 population of A. caninum was reverse transcribed and simultaneously labelled with Cy3 or Cy5 (Agilent). Immediately prior to hybridisation, 500 ng of labelled cRNAs from each of activated and non-activated worms were quantified using a NanoDrop ND-1000 UV-VIS spectrophotometer (NanoDrop) (Agilent), assessed for size distribution and Cy5-dye incorporationquality usingon an Agilent 2100 Bioanalyzer and RNA 6000 LabChip Kit (Agilent) 2100 Bioanalyzer (Agilent 6000 Nano-chip), mixed together and fragmented.

growth_protocol: A. caninum L3s were isolated from the faeces of stray dogs in the greater Brisbane area and surrounding towns in Queensland, Australia, using a standard charcoal coproculture method. Cultures were incubated at ~23°C in a humidified chamber for one week, after which L3s were concentrated using a modified Baermann technique and purified through a nylon filter (20 µm). Larvae were stored for up to four weeks in 50 mM Na2HPO4, 22 mM KH2 PO4, 70 mM NaCl, pH 6.8 in 12.5 cm2 vented tissue culture flasks in the dark at room temperature until use.

extract_protocol: Frozen samples of L3s in Trizol were brought to 4°C and centrifuged (16,000 × g at the same temperature) for 10 min to remove insoluble debris and residual genomic DNA. RNA was then extracted with chloroform, precipitated with isopropanol, washed with absolute ethanol and resuspended in 50 μl of RNAse-free water. Each RNA sample was treated with 2 U of DNase I (Promega) prior to heat denaturation of the enzyme (75 °C for 5 min) and frozen immediately at -80 °C. The integrity of RNA was verified to have an RNA Integrity Number >8.0 using an Agilent 2100 Bioanalyzer and RNA 6000 LabChip Kit 2100 Bioanalyzer (Agilent 6000 Nano-chip (Agilent Technologies). RNA used for microarray analysis was stored as an ethanol precipitate in 75% ethanol at -80°C.

treatment_protocol:
Prior to in vitro activation (serum-stimulation), ensheathed L3s were incubated in 1% HCl for 30 min at ~ 23°C and then resuspended in RPMI-C (RPMI1640 tissue culture medium supplemented with 25 mM HEPES (pH 7.0), 100 IU/ml of penicillin, 100 μg/ml of streptomycin, and 40 μg/ml of gentamycin) (Hawdon et al., 1996). To each well of a 24-well tissue culture plate, 5,000 L3s were added. Activated and non-activated L3 (50,000 of each) were prepared from each of two separate populations of
A. caninum. For the in vitro activation, L3s were incubated overnight at 37°C in 5% CO2; pharyngeal pumping in activated L3s was verified by feeding ~100 of them with FITC-BSA (10 mg/ml) for 3 h and fluorescence was detected using an epi-fluorescence microscope (Hawdon and Schad, 1990). Activated and non-activated L3s were each washed twice in phosphate-buffered saline (PBS, pH 7.4; 23°C) and immediately frozen at -80°C.
Hawdon, J.M., Jones, B.F., Hoffman, D.R., Hotez, P.J., 1996. Cloning and characterization of Ancylostoma-secreted protein. A novel protein associated with the transition to parasitism by infective hookworm larvae. J Biol Chem 271, 6672-6678.

tag: 24-hour, serum-stimulated Ancylostoma caninum L3.

source_name: Non-activated

tag: Ancylostoma caninum L3 incubated under the same conditions as Activated L3 except that serum and GSM were not included in the media.


title: Cy3 vs Cy5 - Activated A. caninum L3, Population III (self hybridization)

data_processing: During extraction, signal intensities were Linear and Lowess-normalized, dye-corrected, and adjusted for local background (Feature extraction software version 9.1.3.1).

tag: 24-hour, serum-stimulated Ancylostoma caninum L3 (Cy3).

tag: 24-hour, serum-stimulated Ancylostoma caninum L3 (Cy5).


title: Activated vs Non-activated A. caninum L3 (Population IV - dye swap)

tag: Incubated exactly as Activated Ancylostoma caninum L3 except that serum and GSM were not added to the media.


title: Cy3 vs Cy5 - Non-activated A. caninum L3, Population IV (self hybridization)

tag: Ancylostoma caninum L3 were incubated exactly as Activated L3 except that serum and GSM were not added to the media. Labelled RNA with Cy3.

tag: Ancylostoma caninum L3 were incubated exactly as Activated L3 except that serum and GSM were not added to the media. Labelled RNA with Cy5.


title: H/W_Liver_vehicle-control_19hr_rep3

hyb_protocol: Hybridization and washing were according to the manufacturer’s instructions (Ambion).  A single labeled miRNA sample was hybridized on to each mirVana miRNA Bioarray. Slides were hybridized at 42ºC for 14 h.

scan_protocol: Arrays were scanned at 10-micron resolution on a PerkinElmer ScanArray microarray scanner (PerkinElmer Life and Analytical Sciences Inc., Waltham, MA) using a photomultiplier tube (PMT) setting of 60 and a laser power of 90.  Each element was located and analyzed using the GenePix Pro 6.0.1.26 software package (Molecular Devices Corporation, Union City, CA).

data_processing: Using LimmaGUI (version 1.8.1; limma, version 2.9.1; (Wettenhall and Smyth, 2004)), the array data were loaded into R statistical package (version 2.4.0), investigated for spatial and distributional homogeneity, background was subtracted, and pre-processed with the variance-stabilizing normalization algorithm (Huber et al., 2002).

source_name: H/W rat Liver vehicle-control 19hr

label_protocol: Two µg of purified small-population RNA from livers of L-E and H/W rats were labeled with a NHS-ester modified Cy5-fluorophore (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) using the mirVana miRNA Labeling Kit (Ambion),  according to the manufacturer’s instructions.

extract_protocol: Total RNA was isolated from rat livers using the mirVana miRNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer’s instructions except flashPAGE fractionation (Ambion) was not performed due to high technical variability. Small-population RNA (<200 nt) was enriched from mirVana-isolated total RNA according to manufacturer’s instructions.  Total RNA yield was quantified by UV spectrophotometry and RNA integrity was verified using an Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA).

treatment_protocol: L-E and H/W rats were treated by gavage with a single dose of 100 µg/kg TCDD or corn oil vehicle for 19 hours

tag: Sex

tag: Treatment

tag: Genotype

tag: Age

tag: Tissue


title: H/W_Liver_TCCD-Treated_19hr_rep3

source_name: H/W rat Liver TCCD-Treated 19hr

value: TCDD 19hr


title: H/W_Liver_TCCD-Treated_19hr_rep4


title: H/W_Liver_vehicle-control_19hr_rep2


title: H/W_Liver_TCCD-Treated_19hr_rep1


title: H/W_Liver_vehicle-control_19hr_rep1


title: H/W_Liver_TCCD-Treated_19hr_rep2


title: Activated versus Non-activated A. caninum L3 (Population III)

treatment_protocol:
Prior to in vitro activation (serum-stimulation), ensheathed L3s were incubated in 1% HCl for 30 min at ~ 23°C and then resuspended in RPMI-C (RPMI1640 tissue culture medium supplemented with 25 mM HEPES (pH 7.0), 100 IU/ml of penicillin, 100 μg/ml of streptomycin, and 40 μg/ml of gentamycin) (Hawdon et al., 1996). To each well of a 24-well tissue culture plate, 5,000 L3s were added. Activated and non-activated L3 (50,000 of each) were prepared from each of two separate populations of A. caninum. For the in vitro activation, L3s were incubated overnight at 37°C in 5% CO2; pharyngeal pumping in activated L3s was verified by feeding ~100 of them with FITC-BSA (10 mg/ml) for 3 h and fluorescence was detected using an epi-fluorescence microscope (Hawdon and Schad, 1990). Activated and non-activated L3s were each washed twice in phosphate-buffered saline (PBS, pH 7.4; 23°C) and immediately frozen at -80°C.
Hawdon, J.M., Jones, B.F., Hoffman, D.R., Hotez, P.J., 1996. Cloning and characterization of Ancylostoma-secreted protein. A novel protein associated with the transition to parasitism by infective hookworm larvae. J Biol Chem 271, 6672-6678.

tag: 24-hour serum stimulated Ancylostoma caninum L3

tag: Ancylostoma caninum L3 (incubated under indentical conditions as (Activated L3 except that serum and GSM were absent).


title: Activated vs Non-activated A. caninum L3 (Population III - Replicate)

tag: 24-hour, serum-stimulated A. caninum L3

treatment_protocol:
Prior to in vitro activation (serum-stimulation), ensheathed L3s were incubated in 1% HCl for 30 min at ~ 23°C and then resuspended in RPMI-C (RPMI1640 tissue culture medium supplemented with 25 mM HEPES (pH 7.0), 100 IU/ml of penicillin, 100 μg/ml of streptomycin, and 40 μg/ml of gentamycin) (Hawdon et al., 1996). To each well of a 24-well tissue culture plate, 5,000 L3s were added. Activated and non-activated L3 (50,000 of each) were prepared from each of two separate populations of A. caninum. For the in vitro activation, L3s were incubated overnight at 37°C in 5% CO2; pharyngeal pumping in activated L3s was verified by feeding ~100 of them with FITC-BSA (10 mg/ml) for 3 h and fluorescence was detected using an epi-fluorescence microscope (Hawdon and Schad, 1990). Activated and non-activated L3s were each washed twice in phosphate-buffered saline (PBS, pH 7.4; 23°C) and immediately frozen at -80°C.
Hawdon, J.M., Jones, B.F., Hoffman, D.R., Hotez, P.J., 1996. Cloning and characterization of Ancylostoma-secreted protein. A
novel protein associated with the transition to parasitism by infective hookworm larvae. J Biol Chem 271, 6672-6678.

tag: Ancylostoma caninum L3 incubated under the same condition as activated L3 except that serum and GSM were not added to the media


title: L-E_Liver_TCCD-Treated_19hr_rep4

source_name: L-E rat Liver TCCD-Treated 19hr

tag: Strain


title: L-E_Liver_TCCD-Treated_19hr_rep3


title: L-E_Liver_vehicle-control_19hr_rep3

source_name: L-E rat Liver vehicle-control 19hr


title: L-E_Liver_vehicle-control_19hr_rep2


title: L-E_Liver_vehicle-control_19hr_rep1


title: L-E_Liver_TCCD-Treated_19hr_rep1


title: L-E_Liver_vehicle-control_19hr_rep4


title: L-E_Liver_TCCD-Treated_19hr_rep2


title: H/W_Liver_vehicle-control_19hr_rep4


title: Activated versus Non-Activated A. caninum L3 (Population III - dye swap)

tag: Ancylostoma L3 incubated under identical conditions as activated L3 except that serum and GSM were not included in the media.


title: Activated vs Non-activated A. caninum L3 (Population III - Replicate - dye swap)

tag: Ancylostoma caninum L3 incubated under the same conditions as Activated L3 except that serum and GSM were not added to the media.

tag: 24-hour serum stimulated Ancylostoma caninum L3.


title: sample 49 positive polarity

status: Public on Aug 14 2007

city: Santa Clara

first: Alicia

last: Scheffer

address: 5301 Stevens Creek Blvd.

institute: Agilent Technologies

description: sample 49 hybridization

hyb_protocol: See manufacturer website: <http://www.chem.agilent.com/scripts/LiteraturePDF.asp?iWHID=52010>

scan_protocol: Scanned on an Agilent G2505A Scanner

data_processing: Agilent Feature Extraction v9.1, default CGH parameters

source_name: sample 49

label_protocol: Agilent DNA labeling kit

extract_protocol: Gentra Puregene

tag: Normal white male from Northern France

tag: Total genomic DNA from blood

source_name: reference DNA 15510

extract_protocol: Unknown

tag: Reference DNA


title: sample 50 positive polarity

description: sample 50 hybridization

source_name: sample 50


title: sample 49 negative polarity rep 4

description: sample 49 dye flip replicate hybridization

extract_protocol: unknown

tag: Reference DNA from Coriell NA15510


title: sample 47 positive polarity

description: sample 47 hybridization

source_name: sample 47


title: sample 49 negative polarity rep 2


title: sample 44 positive poalrity

description: sample 44 hybridization

source_name: sample 44


title: sample 49 negative polarity rep 1


title: sample 46 positive polarity

description: sample 46 hybridization

source_name: sample 46


title: sample 45 positive polarity

description: sample 45 hybridization

source_name: sample 45


title: sample 43 positive polarity

description: sample 43 hybridization

source_name: sample 43


title: sample 37 positive polarity

description: sample 37 hybridization

source_name: sample 37


title: sample 31 positive polarity

description: sample 31 hybridization

source_name: sample 31


title: sample 40 positive polarity

description: sample 40 hybridization

source_name: sample 40


title: sample 42 positive polarity

description: sample 42 hybridization

source_name: sample 42


title: sample 34 positive polarity

description: sample 34 hybridization

source_name: sample 34


title: sample 48 positive polarity

description: sample 48 hybridization

source_name: sample 48


title: self-self A

description: 1 of 3 self self control hybridizations


title: sample 35 positive polarity

description: sample 35 hybridization

source_name: sample 35


title: sample 38 positive polarity

description: sample 38 hybridization

source_name: sample 38


title: sample 41 positive polarity

description: sample 41 hybridization

source_name: sample 41


title: self-self C

description: 3 of 3 self self control hybridizations


title: self-self B

description: 2 of 3 self self control hybridizations


title: sample 49 negative polarity rep 3

source_name: sample 49


title: sample 29 positive polarity

description: sample 29 hybridization

source_name: sample 29

source_name: reference DNA 15510


title: sample 36 positive polarity

description: sample 36 hybridization

source_name: sample 36


title: sample 39 positive polarity

description: sample 39 hybridization

source_name: sample 39


title: sample 30 positive polarity

description: sample 30 hybridization

source_name: sample 30


title: sample 32 positive polarity

description: sample 32 hybridization

source_name: sample 32


title: sample 27 positive polarity

description: sample 27 hybridization

source_name: sample 27


title: sample 28 positive polarity

description: sample 28 hybridization

source_name: sample 28


title: sample 33 positive polarity

description: sample 33 hybridization

source_name: sample 33


title: sample 26 positive polarity

description: sample 26 hybridization

source_name: sample 26


title: sample 24 positive polarity

description: sample 24 hybridization

source_name: sample 24


title: sample 21 positive polarity

description: sample 21 hybridization

source_name: sample 21


title: VA1.32

city: Roslin

first: Michael

middle: Bryan

last: Watson

state: Midlothian, EH25 9PS

address: Roslin Biocentre

department: ARK-Genomics

institute: The Roslin Institute

description: IAH/VLA sample VA1.27

hyb_protocol: Hybridised (4h) according to previously published protocol: 1. Burton, J.E., et al., Development of a multi-pathogen oligonucleotide microarray for detection of Bacillus anthracis. Mol Cell Probes, 2005. 19(5): p. 349-57.

scan_protocol: Slides were scanned using a GenePix Scanner

data_processing: Fluorescence was quantified using GenePix Pro 6.0

organism: Infectious bronchitis virus

source_name: Amplified RNA from cell cultured Avian Infectious Bronchitis Virus (IBV)

label_protocol: Stratascript reverse transcriptase (Stratagene) and random amplification followed by direct labelling with Cy3-dCTP. Primers used:GTT TCC CAG TCA CGA TCN NNN NNN NN and GTT TCC CAG TCA CGA TC

extract_protocol: Total RNA was extracted from cultures of IBV-infected chick kidney cells (RNeasy Mini Kit, Qiagen)


title: sample 23 positive polarity

description: sample 23 hybridization

source_name: sample 23


title: VA1.24

description: IAH/VLA sample VA1.24

tag: Amplified RNA from cell cultured Avian Infectious Bronchitis Virus (IBV)


title: sample 25 positive polarity

description: sample 25 hybridization

source_name: sample 25


title: VA1.27

description: IAH/VLA sample VA1.27


title: sample 17 positive poalrity

description: sample 17 hybridization

source_name: sample 17


title: VA1.30

description: IAH/VLA sample VA1.27


title: sample 14 positive poalrity

description: sample 14 hybridization

source_name: sample 14


title: VA1.11

description: IAH/VLA sample VA1.11

hyb_protocol: Hybridised (2h) according to previously published protocol: 1. Burton, J.E., et al., Development of a multi-pathogen oligonucleotide microarray for detection of Bacillus anthracis. Mol Cell Probes, 2005. 19(5): p. 349-57.

scan_protocol: Slides were scanned using a Hewlett Packard ScanArray Lite

data_processing: Fluorescence was quantified using ScanArray Express 2.0.

organism: Foot-and-mouth disease virus A

source_name: Amplified RNA from cell cultured FMDV type A

label_protocol: MultiScribe reverse transcriptase (Applied Biosystems) and random amplification followed by direct labelling with Cy3-dCTP. Primers used:GTT TCC CAG TCA CGA TCN NNN NNN NN and GTT TCC CAG TCA CGA TC

extract_protocol: Total RNA was extracted from FMD virus cultured in primary bovine thyroid cells (Rneasy Mini Kit, Qiagen)


title: VA1.7

description: IAH/VLA sample VA1.7

hyb_protocol: Hybridised according to previously published protocol: 1. Burton, J.E., et al., Development of a multi-pathogen oligonucleotide microarray for detection of Bacillus anthracis. Mol Cell Probes, 2005. 19(5): p. 349-57.

tag: Amplified RNA from cell cultured FMDV type A


title: sample 07 positive polarity

description: sample 07 hybridization

source_name: sample 07

source_name: reference DNA 15510


title: sample 18 positive polarity

description: sample 18 hybridization

source_name: sample 18


title: VA1.9

description: IAH/VLA sample VA1.9

organism: Foot-and-mouth disease virus O

source_name: Amplified RNA from sheep infected with FMDV type O

label_protocol: MultiScribe reverse transcriptase (Applied Biosystems)  and random amplification followed by direct labelling with Cy3-dCTP. Primers used:GTT TCC CAG TCA CGA TCN NNN NNN NN and GTT TCC CAG TCA CGA TC

extract_protocol: Total RNA was extracted from epithelium of sheep 2 days post infection with O UKG 35/2001 (MagNa Pure LC Total Nucleic Acid Isolation Kit Roche)


title: sample 15 positive polarity

description: sample 15 hybridization

source_name: sample 15


title: sample 22 positive polarity

description: sample 22 hybridization

source_name: sample 22


title: sample 10 positive polarity

description: sample 10 hybridization

source_name: sample 10


title: sample 19 positive poalrity

description: sample 19 hybridization

source_name: sample 19


title: sample 12 positvie polarity

description: sample 12 hybridization

source_name: sample 12


title: sample 16 positive polarity

description: sample 16 hybridization

source_name: sample 16


title: sample 11 positive polarity

description: sample 11 hybridization

source_name: sample 11


title: sample 20 positive poalrity

description: sample 20 hybridization

source_name: sample 20


title: VA1.5

description: IAH/VLA sample VA1.5

source_name: Amplified RNA from cell cultured FMDV type O


title: VA1.6

description: IAH/VLA sample VA1.6


title: sample 01 positive polarity

description: sample 01 hybridization

source_name: sample 01


title: sample 13 positive poalrity

description: sample 13 hybridization

source_name: sample 13


title: VA1.4

description: IAH/VLA sample VA1.4

tag: Amplified RNA from cell cultured FMDV type O


title: sample 08 positive polarity

description: sample 08 hybridization

source_name: sample 08


title: VA1.3

description: IAH/VLA sample VA1.3


title: sample 03 positive polarity

description: sample 03 hybridization

source_name: sample 03

source_name: refernce DNA 15510


title: sample 05 positive polarity

description: sample 05 hybridization

source_name: sample 05

source_name: reference DNA 15510


title: sample 06 positive polarity

description: sample 06 hybridization

source_name: sample 06


title: sample 09 positive polarity

description: sample 09 hybridization

source_name: sample 09


title: sample 04 positive poalrity

description: sample 04 hybridization

source_name: sample 04


title: sample 02 positive polarity

description: sample 02 hybridization

source_name: sample 02