title: Pure_Dehalo_ethenogenes_195_DNA_PosCtrl_Rep1

type: genomic

city: Berkeley

first: Kimberlee

last: West

address: 207 O'Brien Hall

department: Civil and Environmental Engineering

institute: University of California, Berkeley

description: Positive control probe sets target Bacillus subtilis genes lys, phe, thr, and dap. To generate control DNA, plasmids containing lys, phe, thr, and dap were purified from strains ATCC87482, ATCC87483, ATCC87484, and ATCC87486, respectively. These plasmid standards were spiked into each sample before fragmentation. Negative control probe sets were designed to hybridize with specific Escherichia coli and bacteriophage genes that were not expected to be present in experimental cultures.

hyb_protocol: gDNA was prepared for application to microarrays with minimal modifications to the protocols outlined in section 3 of the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA).  Microarray chips were prehybridized with 1 x hybridization buffer [100 mM MES (Sigma-Aldrich), 1 M [Na+] (Sigma-Aldrich), 20 mM EDTA (Sigma-Aldrich), 0.01% Surfact-Amps Tween-20 (Pierce Chemical)] in a chip-rotisserie oven for at least 20 minutes at 60 rmp and 45°C. The samples were denatured by heating to 99°C for 5 minutes. Hybridization buffer was removed from each microarray chip and replaced with an equal volume of sample hybridization mixture (1 x MES hybridization buffer, 7.8 % DMSO [Sigma-Aldrich], 0.1 mg/ml herring sperm DNA [Promega Corporation], 0.5 mg/ml BSA [Invitrogen Life Technologies, Carlsbad, CA], 50 pM B2 control oligonucleotide [Affymetrix], and 1 µg fragmented labeled DNA). Hybridization was carried out by incubating in a chip-rotisserie oven (Affymetrix) for 16 hours at 45°C and 60 rmp. Chips were washed and stained according to the Affymetrix protocol using the Affymetrix GeneChip fluidics station.

scan_protocol: Affymetrix chips were scanned according to the protocols outlined in section 3, chapter 3 of the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA).

data_processing: The hybridization signal intensities for each probe set was computed using Affymetrix GeneChip Operating Software and the MAS5 algorithm. The data set of each microarray was normalized by scaling the signal intensities of the added positive controls to a target signal intensity of 2500 and adjusting all other probe set signal intensities accordingly with respect to the scaled positive control signal intensities, thus allowing for comparisons between microarray chips.

molecule: genomic DNA

organism: Dehalococcoides mccartyi 195

label_protocol: gDNA was prepared for application to microarrays with minimal modifications to the protocols outlined in section 3 of the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA).  Briefly, purified gDNA (1 μg per array) was mixed with the positive control plasmids (7.6 nM lys, 15.2 nM phe, 30.4 nM thr, and 114.0 nM dap) described above and fragmented to an average size of 50 to 200 bp by enzymatic digestion with amplification grade DNase I (Invitrogen Life Technologies, Carlsbad, CA). Each 50-μl fragmentation reaction consisted of gDNA and 0.02 units DNase I per μg DNA diluted in 1 x One-Phor-All Buffer (Amersham Biosciences).  The mixture was incubated for 20 minutes at 25°C, followed by 10 minutes at 98°C to inactivate the DNase I. The fragmentation products were visualized on a 4% MetaPhor Agarose (Cambrex Bio Science Rockland, Inc., Rockland, ME) electrophoresis gel run at 70 V for 2 hours. 25 µl of the fragmentation product was then used for further processing. The fragmentation products were biotin end-labeled by adding 25 µl of fragmentation product to 10 µl of 5 x reaction buffer (Affymetrix, Santa Clara, CA), 2 µl 7.5 mM GeneChip DNA labeling reagent (Affymetrix), 2 µl terminal deoxynucleotidyl transferase (Promega), and 11 µl nuclease-free water, incubating at 37°C for 60 minutes, and adding 2 µl 0.5 M EDTA (pH 8 .0) (Invitrogen Life Technologies, Carlsbad, CA) to stop the reaction.

growth_protocol: Dehalococcoides ethenogenes 195 was grown in 100-ml batch pure cultures with a defined mineral salts medium containing 5mM acetate as the carbon source, H2 as the electron donor (added as H2:CO2 gas [80:20 vol:vol]), and 78 μmol TCE as the electron acceptor.

extract_protocol: Dehalococcoides ethenogenes 195 cells were harvested from ten 100-ml batches per culture to assure that a sufficient quantity of DNA could be extracted for application to microarrays. Cells were collected by vacuum filtration with hydrophilic Durapore membrane filters (0.22 µm pore size, 47 mm diameter [Millipores, Billerica, MA]), and the filters were stored in 2-ml microcentrifuge tubes at –80°C until further processing. gDNA was isolated from filters using a phenol extraction method as follows. The 2-ml microcentrifuge tubes containing frozen filters were amended with 250 µl lysis buffer (200 mM Tris [pH 8.0], 50 mMEDTA, and 200 mM NaCl), 100 µl 10% sodium dodecyl sulfate, 1 g 100-µm-diameter zirconia-silica beads (Biospec Products, Bartlesville, OK), and 1.0 ml buffer-equilibrated phenol (pH 8.0) (Sigma-Aldrich, St. Louis, MO). Cells were lysed by heating the tubes to 65°C for 2 min, bead beating them with a Mini Bead Beater (Biospec Products) for 2 min, incubating for 8 min at 65°C, and bead beating again for an additional 2 min. Cellular debris was collected by centrifugation (12,000 x g for 5 minutes at 4°C), and the aqueous lysate was transferred to a new DNase-free microcentrifuge tube. The aqueous lysate was extracted twice with 1 volume of phenol (pH 8.0)-chloroform-isoamylalcohol (125:24:1 vol:vol) and once with 1 volume of chloroform-isoamylalcohol (24:1 vol:vol) (Sigma-Aldrich). DNA was precipitated by adding 0.1 volume of 3 M ammonium acetate (pH 5.2) and 1 volume of 100% isopropanol. The precipitate was collected by centrifugation, washed once with 80% ethanol, and resuspended in 40 µl of nuclease-free water. Contaminating RNA was removed by RNase digestion with DNase-free RNase according to the manufacturer’s instructions (Roche Applied Science, Indianapolis, IN). The purified DNA was stored at –80°C prior to further use.

tag: Pure culture Dehalococcoides ethenogenes 195 collected in March 2006 were used as a positive control for the experiment.


title: Pure_Dehalobacter_restrictus_DNA_NegCtrl_Rep1

status: Public on Sep 01 2008

state: CA

country: USA

label: biotin

organism: Dehalobacter restrictus

source_name: Pure culture Dehalobacter restrictus

growth_protocol: Dehalobacter restrictus PER-K23 (DSMZ strain number 9455) was grown in 100-ml batch pure cultures with a defined mineral salts medium containing 5mM acetate as the carbon source, H2 as the electron donor (added as H2:CO2 gas [80:20 vol:vol]), and 78 μmol TCE as the electron acceptor.

extract_protocol: Dehalobacter restrictus cells were harvested from ten 100-ml batches per culture to assure that a sufficient quantity of DNA could be extracted for application to microarrays. Cells were collected by vacuum filtration with hydrophilic Durapore membrane filters (0.22 µm pore size, 47 mm diameter [Millipores, Billerica, MA]), and the filters were stored in 2-ml microcentrifuge tubes at –80°C until further processing. gDNA was isolated from filters using a phenol extraction method as follows. The 2-ml microcentrifuge tubes containing frozen filters were amended with 250 µl lysis buffer (200 mM Tris [pH 8.0], 50 mMEDTA, and 200 mM NaCl), 100 µl 10% sodium dodecyl sulfate, 1 g 100-µm-diameter zirconia-silica beads (Biospec Products, Bartlesville, OK), and 1.0 ml buffer-equilibrated phenol (pH 8.0) (Sigma-Aldrich, St. Louis, MO). Cells were lysed by heating the tubes to 65°C for 2 min, bead beating them with a Mini Bead Beater (Biospec Products) for 2 min, incubating for 8 min at 65°C, and bead beating again for an additional 2 min. Cellular debris was collected by centrifugation (12,000 x g for 5 minutes at 4°C), and the aqueous lysate was transferred to a new DNase-free microcentrifuge tube. The aqueous lysate was extracted twice with 1 volume of phenol (pH 8.0)-chloroform-isoamylalcohol (125:24:1 vol:vol) and once with 1 volume of chloroform-isoamylalcohol (24:1 vol:vol) (Sigma-Aldrich). DNA was precipitated by adding 0.1 volume of 3 M ammonium acetate (pH 5.2) and 1 volume of 100% isopropanol. The precipitate was collected by centrifugation, washed once with 80% ethanol, and resuspended in 40 µl of nuclease-free water. Contaminating RNA was removed by RNase digestion with DNase-free RNase according to the manufacturer’s instructions (Roche Applied Science, Indianapolis, IN). The purified DNA was stored at –80°C prior to further use.

tag: Pure culture Dehalobacter restrictus collected in March 2006 was used as a negative control for the experiment.


title: Dechlorinating_Community_DNA_ANAS_Rep5

organism: Dehalococcoides

source_name: Dehalococcoides-containing Enrichment Community ANAS

growth_protocol: The Dehalococcoides-containing enrichment culture is grown and maintained in an anaerobic semi-batch reactor supplemented with modified-Tanner medium. Lactate (25 mM final concentration) is supplied as both the carbon source and electron donor, while TCE (0.1 mM final concentration) is supplied as the terminal electron acceptor.

extract_protocol: A 50-ml sample of the ANAS culture was collected after one 100- μM dose of TCE was degraded to VC and ethene. Cells were collected by centrifugation (12,000 x g for 3 minutes at 4°C), the supernatants were discarded, and the cell pellets were stored at –80°C until processing. ANAS genomic DNA (gDNA) was isolated from frozen cell pellets using an UltraClean Mega Prep Soil DNA kit (Mo Bio Laboratories, Carlsbad, CA) according to the manufacturer's instructions. DNA was stored at –80°C prior to further use.

tag: An anaerobic microbial consortium (referred to as ANAS) was enriched from contaminated soil obtained from Alameda Naval Air Station and has been functionally stable for over 7 years with the ability to reductively dechlorinate TCE completely to ethene. Previous clone library studies have shown that Dehalococcoides bacteria comprise approximately 30% of the ANAS culture.Sample "Dechlorinating_Community_DNA_ANAS_Rep5" was collected from the ANAS culture in March 2007.


title: Dechlorinating_Community_DNA_ANAS_Rep2

tag: An anaerobic microbial consortium (referred to as ANAS) was enriched from contaminated soil obtained from Alameda Naval Air Station and has been functionally stable for over 7 years with the ability to reductively dechlorinate TCE completely to ethene. Previous clone library studies have shown that Dehalococcoides bacteria comprise approximately 30% of the ANAS culture. Sample "Dechlorinating_Community_DNA_ANAS_Rep2" was collected from the culture in March 2006.


title: Dechlorinating_Community_DNA_ANAS_Rep1

tag: An anaerobic microbial consortium (referred to as ANAS) was enriched from contaminated soil obtained from Alameda Naval Air Station and has been functionally stable for over 7 years with the ability to reductively dechlorinate TCE completely to ethene. Previous clone library studies have shown that Dehalococcoides bacteria comprise approximately 30% of the ANAS culture. Sample "Dechlorinating_Community_DNA_ANAS_Rep1" was collected from the culture in March 2006.


title: Pure_Dehalobacter_restrictus_DNA_NegCtrl_Rep2


title: Pure_Dehalo_ethenogenes_195_DNA_PosCtrl_Rep2

source_name: Pure culture Dehalococcoides ethenogenes 195


title: Dechlorinating_Community_DNA_ANAS_Rep4

tag: An anaerobic microbial consortium (referred to as ANAS) was enriched from contaminated soil obtained from Alameda Naval Air Station and has been functionally stable for over 7 years with the ability to reductively dechlorinate TCE completely to ethene. Previous clone library studies have shown that Dehalococcoides bacteria comprise approximately 30% of the ANAS culture.Sample "Dechlorinating_Community_DNA_ANAS_Rep4" was collected from the ANAS culture in March 2007.


title: ATF2 WT-1

type: RNA

city: Bethesda

first: Sean

last: Davis

state: MD

address: 37 Convent Drive, Room 6138

institute: National Cancer Institute

description: Technical replicate #1 of ATF2 wildtype. These mice are mixed background of FVB and C57bl6 and the age when they got the papillomas was about 9 months.

hyb_protocol: Hybridization was per Illumina standard protocol

scan_protocol: Images were scanned on an Illumina BeadStation scanner

data_processing: Data were extracted using BeadStudio version 3 without any normalization and were then processed after log2 transformation using quantile normalization.

molecule: total RNA

organism: Mus musculus

source_name: RNA was prepared from papilomas of 2-3 mice (2 for WT and 3 for Knockout) and this was pooled to perform the array. Each pooled sample was run in triplicates.   The mice were treated once with DMBA, followed by TPA application for 30 wks.

label_protocol: Labeling was per Illumina standard protocol.

tag: ATF2 Genotype

value: WT


title: Dechlorinating_Community_DNA_ANAS_Rep3

tag: An anaerobic microbial consortium (referred to as ANAS) was enriched from contaminated soil obtained from Alameda Naval Air Station and has been functionally stable for over 7 years with the ability to reductively dechlorinate TCE completely to ethene. Previous clone library studies have shown that Dehalococcoides bacteria comprise approximately 30% of the ANAS culture. Sample "Dechlorinating_Community_DNA_ANAS_Rep3" was collected from the culture in March 2007.


title: T4-2_vector_rep3

city: San Francisco

first: Kelvin

middle: Kun-Chih

last: Tsai

address: 513 Parnassus Avenue, Rm S1364

department: Surgery

institute: University of California, San Francisco

description: Please refer to Affymetrix Expression Analysis Technical Manual (http://www.affymetrix.com/support/technical/manual/expression_manual.affx) for detailed experimental procedures.

hyb_protocol: Heat the following mixture at 99 degrees for 5 min: Control Oligonucleotide B2 (3nM) 5 ul, 20X Eukaryotic Hybridization controls 15 ul, Herring Sperm DNA (10mg/mL) 3 ul, BSA (50mg/ml) 3 ul, 2X Hybridization Buffer 150 ul, DMSO 30 ul, H2O to final volume of 300 ul.  Moisten array with 200 ul 1X hybridization buffer and then place tough spots on and incubate in rotating oven for 10 minutes at 45 degrees. Transfer the cRNA hyb mix to the 45 degree oven to cool down for 5 minutes. Spin down tube at max speed in a microcentrifuge for 5 minutes. Remove the  1X hyb buffer from chips carefully and carefully load the spun-down samples (250 ul) onto chip. Reapply new tough spots. Place chips in rotating oven O/N.

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

data_processing: The hybridization intensity data was processed using the GeneChip Operating software (Affymetrix). Affymetrix .cel files (probe intensity files) were processed with ArrayAssist Lite (v3.4, Stratagene). The files were imported and processed with the GC-RMA algorithm to yield probe set intensities and additionally, Affymetrix Preset, Absent, Marginal flags were computed. These values were exported in .chp files, which were subsequently imported into the Partek Genomics Suite software (v6.2, Partek).

organism: Homo sapiens

source_name: T4-2 breast epithelial cells expressing an empty retrovial vector

label_protocol: Twenty microgram of total RNA from each sample was processed to produce biotinylated cRNA targets.

growth_protocol: Cells were propagated in chemically defined H14 medium (V. M. Weaver et al., J. Cell Biol. 137, 231 (1997)) for 7 days, after which they were cultured on top of reconstituted basement membrane (Matrigel, BD Biosciences) for 6 days (G. Y. Lee et al., Nat Method 4, 359 (2007)).

extract_protocol: Total RNA was extracted from monolayer culture or 3D culture by a modified TRI reagent procedure (P. Chomczynski et al., Biotechniques 19, 942 (1995)) and purified using an RNeasy Mini Kit (Qiagen).

treatment_protocol: T4-2 cells carrying pMFG-tet-GFP and stably express GFP by retrovirus-mediated gene transduction.

tag: HMT3522 T4-2 neoplastic breast epithelial cells derived from epidermal growth factor withdrawal (P. Briand et al., Cancer Res 56, 2039 (1996)).


title: T4-2_vector_rep2


title: PrrA-PpsR-_DDMSO_1

status: Public on Aug 31 2008

city: Houston

first: patrice

last: bruscella

state: TX

address: 6431 Fannin

department: Microbiology and Molecular Genetics

institute: University of Texas Health Science Center

description: Anaerobic dark DMSO conditions

hyb_protocol: According to Affymetrix protocol

scan_protocol: According to Affymetrix protocol

data_processing: Scanned image was analyzed using the Affymetrix Microarray Suite 5.0 (MAS5, Affymetrix Inc.) to obtain signal value, detection calls (present/marginal/absent) and p values

organism: Cereibacter sphaeroides 2.4.1

source_name: Rhodobacter sphaeroides PrrA-PpsR-

label_protocol: According to Affymetrix protocol

extract_protocol: According to Roh et al., 2004

tag: Anaerobic dark DMSO conditions:

tag: Sparging with 95% N2, 5% CO2 in the dark

tag: Sistrom minimal medium A supplemented with final concentration of 0.1% YE and 0.5% DMSO


title: PrrA-PpsR-_DDMSO_2

description: Anaerobic dark DMSO conditions


title: PrrA-PpsR-_DDMSO_3

description: Anaerobic dark DMSO conditions


title: shLoxl2_1 CY3

status: Public on Aug 29 2008

city: Madrid

first: Gema

last: Moreno-Bueno

state: Madrid

address: Arturo Duperier

department: Biochemistry

country: Spain

institute: IIB

description: After hybridization and washing, slides were scanned in an Axon GenePix Scanner (Axon Instruments, Inc., Union City, CA) and analyzed using Feature Extraction Software 6.1.1 (Agilent).

hyb_protocol: Labelling and hybridization were performed using Low RNA Linear Amplification Kit and In Situ Hybridization Kit Plus (Agilent), respectively, following manufacturer’s protocol.

data_processing: The unsupervised analyses were visualized using the SOTA and TreeView software assuming euclidean distances between genes (http://bioinfo.cnio.es/cgi-bin/tools/clustering/sotarray).

source_name: shLoxl2 CY3

extract_protocol: RNA was isolated using RNAesy Extraction Kit (QIAGen).

tag: Haca cells shLoxl2 labelled with cy3 dye

source_name: control cell line shGFP CY5

tag: Haca cells shGFP labelled with cy5 dye


title: RBP2 siRNA, biol. repl. 2

city: Chicago

first: Elizaveta

middle: V

last: Benevolenskaya

state: IL

address: 900 S. Ashland Ave.

department: Biochemistry and Molecular Genetics

institute: University of Illinois at Chicago

description: gene expression data from SAOS-2 cells treated with RBP2 siRNA 4

hyb_protocol: The fragmented cRNA was added to a hybridization solution containing several biotinylated controls and hybridized to the microarray overnight at 45C

scan_protocol: Each cRNA bound at its complementary oligonucleotide is excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions are captured. These measures provide the basis of subsequent biostatistical analysis. dChip computes its own Model-Based Expression Indices (MBEIs) directly from the .CEL files. We download all .CEL files into a common directory. If the .TXT files are included in the same directory, dChip will use the presence calls calculated by MAS 5.0.

data_processing: The data were analyzed with dChip using default analysis settings. The chip with median overall brightness is chosen, and all other arrays are normalized to it. dChip software, designed by Li and Wong 2001 (Model-based analysis of oligonucleotide arrays: Expression index computation and outlier detection. Proc. Natl. Acad. Sci. Vol. 98, 31-36) provides model-based estimates of expression levels of genes

source_name: transfected SAOS-2 cells

label_protocol: RNA was converted into cDNA and second strand cDNA synthesis was then carried out. The double-stranded cDNA was used as the template in an in vitro transcription reaction containing biotinylated CTP and UTP. The biotinylated complementary RNA (cRNA) was fragmented and purified.

growth_protocol: SAOS-2 cells were transfected using Lipofectamine 2000 (Invitrogen) and placed under selection with 0.6 μg/ml puromycin. Total RNA isolation was carried out on the fifth day after transfection for samples RBP2siRNA and RBP2scsiRNA and on the third day for samples RB.

extract_protocol: RNA was extracted using the NucleoSpin RNA kit (Clontech) according to the manufacturer's instructions.

treatment_protocol: SAOS-2 were transfected with 1 μg of pBabePuro plasmid and 40 nM siRNA 4sc for RBP2scsiRNA samples or siRNA 4 for RBP2siRNA samples; SAOS-2 were transfected with 1 μg of pBabePuro-HA-RB(379-928) plasmid and 40 nM siRNA 4sc for RB sample. Both transfections and RNA isolations were done separately for each set of three samples (from 1 to 3).

tag: cells with RBP2 knockdown


title: scrambled siRNA, biol. repl. 2

description: gene expression data from SAOS-2 cells treated with control siRNA 4

tag: control cells


title: shSNAIL_1 CY5

description: Labelling and hybridization were performed using Low RNA Linear Amplification Kit and In Situ Hybridization Kit Plus (Agilent), respectively, following manufacturer’s protocol.

scan_protocol: After hybridization and washing, slides were scanned in an Axon GenePix Scanner (Axon Instruments, Inc., Union City, CA) and analyzed using Feature Extraction Software 6.1.1 (Agilent).

source_name: shSNAIL_1 CY5

tag: Haca cells shSNAIL labelled with cy5 dye

source_name: control cell line shGFP CY3

tag: Haca cells shGFP labelled with cy3 dye


title: RBP2 siRNA, biol. repl. 1


title: shLoxl2_1 CY5

data_processing: analyzed using Feature Extraction Software 6.1.1 (Agilent).

source_name: shLoxl2 CY5

label_protocol: Labelling and hybridization were performed using Low RNA Linear Amplification Kit and In Situ Hybridization Kit Plus (Agilent), respectively, following manufacturer’s protocol.

tag: Haca cells shLoxl2 labelled with cy5 dye


title: T4-2_vector_rep1


title: pRB overexp., biol. repl. 2

description: gene expression data from SAOS-2 cells transfected with pRB expression plasmid

tag: cells with pRB overexpression


title: shLoxl2_2 CY3

source_name: shLoxl2_2 CY3


title: shSNAIL_2 CY5

source_name: shSNAIL_2 CY5


title: scrambled siRNA, biol. repl. 3


title: pRB overexp., biol. repl. 3


title: RBP2 siRNA, biol. repl. 3


title: pRB overexp., biol. repl. 1


title: Earthworm exposed to RDX_Rep2 vs TNT_Rep3

status: Public on Aug 28 2008

city: Vicksburg

first: Ping

last: Gong

state: MS

address: 3909 Halls Ferry Road

department: Environmental Laboratory

institute: US Army ERDC

description: A RNAlater-ICE fixed worm was chopped into 8-10 pieces in equal length. Total RNA was isolated using Qiagen RNeasy Mini Kit from each of the 8-10 pieces and was pooled as one biological replicate representing each individual earthworm. The pooled total RNA was purified to obtain mRNA using NucleoTrap Nucleic Acid Purification Kit (BD Biosciences).

hyb_protocol: Labeled cDNA probes for both channels (Genisphere) were added to the array, covered with a glass cover slip, which was then placed in a 50-ml tube. A few drops of water was also added into the tube to keep the air moisture. The capped tube was incubated at 55oC in a hybridization oven without rotation overnight (ca. 16 hr). Arrays were post-hyb washed according to manufacture's instruction (Genisphere).

scan_protocol: Arrays were scanned at 5-um resolution using VersArray ChipReader (Bio-Rad).

data_processing: A spot was flagged out if its raw signal intensity was below its background level or the mean + 2x standard deviation of the 256 blank spots. The filtered spot intensity data were normalized by (1) subtraction of background intensity, and (2) centering to the mean spot signal intensity of each channel.

molecule: polyA RNA

organism: Eisenia fetida

source_name: mRNA from worm (Rep#2) treated with 30 ppm of RDX labeled with Cyanine-3 (green)

label_protocol: 30 ng mRNA was reverse-transcribed into cDNA and labeled using Genisphere 3DNA 900 Expression Array Detection Cy3 kit.

extract_protocol: RNeasy kit (Qiagen) and NucleoTrap Nucleic Acid Purification Kit (BD Biosciences)

tag: Adult earthowrms exposed in soil for 28 days.

tag: Worms were snap frozen in liquid nitrogen.

tag: Worm tissue was fixed in RNAlater-ICE (Ambion).

source_name: mRNA from worm (Rep#3) treated with 50 ppm of TNT labeled with Alex Fluor 647 (red)

label_protocol: 30 ng mRNA was reverse transcribed into cDNA and labeled using Genisphere 3DNA 900 Expression Array Detection Alex Fluor 647 kit.

tag: Worm tissue was fixed in RNAlater-ICe (Ambion).


title: scrambled siRNA, biol. repl. 1


title: Earthworm exposed to TNT+RDX_Rep4 vs Control_Rep1

source_name: mRNA from worm (Rep#4) treated with 50 ppm of TNT and 30 ppm of RDX labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#1) treated with solvent control soil labeled with Alex Fluor 647 (red)


title: Earthworm exposed to RDX_Rep3 vs TNT_Rep4

source_name: mRNA from worm (Rep#3) treated with 30 ppm of RDX labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#4) treated with 50 ppm of TNT labeled with Alex Fluor 647 (red)


title: Earthworm exposed to TNT_Rep1 vs TNT+RDX_Rep2

source_name: mRNA from worm (Rep#1) treated with 50 ppm of TNT labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#2) treated with 50 ppm of TNT and 30 ppm of RDX labeled with Alex Fluor 647 (red)


title: Earthworm exposed to Control_Rep5 vs RDX_Rep1

source_name: mRNA from worm (Rep#5) treated with solvent control soil labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#1) treated with 30 ppm of RDX labeled with Alex Fluor 647 (red)


title: shLoxl2_2 CY5

source_name: shLOXl2_2 CY5


title: Earthworm exposed to Control_Rep4 vs RDX_Rep5

source_name: mRNA from worm (Rep#4) treated with solvent control soil labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#5) treated with 30 ppm of RDX labeled with Alex Fluor 647 (red)


title: Earthworm exposed to TNT+RDX_Rep5 vs Control_Rep2

source_name: mRNA from worm (Rep#5) treated with 50 ppm of TNT and 30 ppm of RDX labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#2) treated with solvent control soil labeled with Alex Fluor 647 (red)


title: Earthworm exposed to Control_Rep2 vs RDX_Rep3

source_name: mRNA from worm (Rep#2) treated with solvent control soil labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#3) treated with 30 ppm of RDX labeled with Alex Fluor 647 (red)


title: Earthworm exposed to Control_Rep5 vs RDX_Rep5


title: Earthworm exposed to Control_Rep3 vs RDX_Rep4

source_name: mRNA from worm (Rep#3) treated with solvent control soil labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#4) treated with 30 ppm of RDX labeled with Alex Fluor 647 (red)


title: Earthworm exposed to TNT_Rep5 vs TNT+RDX_Rep1

source_name: mRNA from worm (Rep#5) treated with 50 ppm of TNT labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#1) treated with 50 ppm of TNT and 30 ppm of RDX labeled with Alex Fluor 647 (red)


title: Earthworm exposed to TNT+RDX_Rep3 vs Control_Rep5

source_name: mRNA from worm (Rep#3) treated with 50 ppm of TNT and 30 ppm of RDX labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#5) treated with solvent control soil labeled with Alex Fluor 647 (red)


title: Earthworm exposed to Control_Rep2 vs RDX_Rep2

source_name: mRNA from worm (Rep#2) treated with 30 ppm of RDX labeled with Alex Fluor 647 (red)


title: Earthworm exposed to Control_Rep1 vs RDX_Rep2

source_name: mRNA from worm (Rep#1) treated with solvent control soil labeled with Cyanine-3 (green)


title: Earthworm exposed to Control_Rep3 vs RDX_Rep3


title: Earthworm exposed to TNT+RDX_Rep3 vs Control_Rep4

source_name: mRNA from worm (Rep#4) treated with solvent control soil labeled with Alex Fluor 647 (red)


title: Earthworm exposed to Control_Rep1 vs RDX_Rep1

source_name: mRNA from worm (Rep#1) treated with 30 ppm of RDX labeled with Alex Fluor 647 (red)


title: Earthworm exposed to TNT+RDX_Rep1 vs Control_Rep2

source_name: mRNA from worm (Rep#1) treated with 50 ppm of TNT and 30 ppm of RDX labeled with Cyanine-3 (green)


title: Earthworm exposed to TNT+RDX_Rep5 vs Control_Rep1

source_name: mRNA from worm (Rep#1) treated with solvent control soil labeled with Alex Fluor 647 (red)


title: Earthworm exposed to TNT+RDX_Rep2 vs Control_Rep4

source_name: mRNA from worm (Rep#2) treated with 50 ppm of TNT and 30 ppm of RDX labeled with Cyanine-3 (green)


title: Earthworm exposed to TNT+RDX_Rep4 vs Control_Rep5

source_name: mRNA from worm (Rep#4) treated with 50 ppm of TNT and 30 ppm of RDX labeled with Cyanine-3 (green)


title: Earthworm exposed to RDX_Rep5 vs TNT_Rep1

source_name: mRNA from worm (Rep#5) treated with 30 ppm of RDX labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#1) treated with 50 ppm of TNT labeled with Alex Fluor 647 (red)


title: Earthworm exposed to TNT+RDX_Rep1 vs Control_Rep3

source_name: mRNA from worm (Rep#3) treated with solvent control soil labeled with Alex Fluor 647 (red)


title: Earthworm exposed to RDX_Rep4 vs TNT_Rep5

source_name: mRNA from worm (Rep#4) treated with 30 ppm of RDX labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#5) treated with 50 ppm of TNT labeled with Alex Fluor 647 (red)


title: C57BL/6J mice on folate depletion diet for 14 days then 7 days on control, both with 50mM saccharine (BioRep2,TechRep)

status: Public on Aug 27 2008

city: Cleveleand

first: Joseph

last: Nadeau

state: OH

address: 10900 Euclid Avenue

department: Genetics

institute: Case Western Reserve University

description: No additional information

hyb_protocol: THE INSTITUTE FOR GENOMIC RESEARCHStandard Operating Procedure TITLE: MICROARRAY LABELED PROBE HYBRIDIZATION PAGE: 1 of 5 SOP #: M005 REVISION LEVEL: 3 EFFECTIVE DATE: 9/11/02 AUTHOR:Jeremy Hasseman PRIMARY REVIEWERS:Emily Chen, Ivana Yang 1. PURPOSE This protocol describes the hybridization of a Cy labeled cDNA probe (mix of Cy3 and Cy5) onto coated slide spotted with PCR amplified cDNA. 2. SCOPE This procedural format is currently utilized by Human Colon Cancer and Mouse microarray projects under the supervision of John Quackenbush within the Mammalian Genomics Dept. 3. MATERIALS 3.1 20X Saline-Sodium Citrate (SSC) (Sigma; Cat # S-6639) 3.2 10% Sodium Dodecyl Sulfate (SDS)(Life Technologies; Cat # 15553-035) 3.3 Bovine Serum Albumin (BSA) (Sigma; Cat # A-9418) 3.4 Formamide, redistilled (Life Technologies; Cat # 15515-081) 3.5 Isopropanol (Fisher Scientific; Cat # A451-1) 3.6 Coplin jar (VWR; Cat # 25457-200) 3.7 Human COT1-DNA (Life Technologies; Cat # 15279-011) 3.8 Mouse COT1-DNA (Life Technologies; Cat # 18440-016) 3.9 Poly(A)-DNA (Pharmacia; Cat # 27-7836-01) 3.10 Microscope Cover Glass (Fisher Scientific; Cat # 12-545J) 3.11 Corning UltraGAPSä Coated Slides (bar-coded) (Corning; Cat # 40015) 3.12 Hybridization chamber (Corning Costar; Cat #2551) 3.13 1 L .22 mm CA (cellulose acetate) Filter System (Corning; Cat #430517) 3.14 Pressurized air duster (Fellowes; Cat # 99790) or clean in-house pressurized air source 4. PROCEDURE 4.1 UV Cross-linking and Prehybridizaton 4.1.1 Aminosilane coated slides (Corning UltraGAPSä Coated Slides) spotted with cDNA in 50% DMSO are UV cross-linked at ~120 mJ. 4.1.2 Prepare prehybridization buffer (5X SSC, 0.1% SDS, 1% BSA) and sterilize by filtration using a CA filter. Preheat at 42oC for ~30 minutes before use. 4.1.3 Place the printed slide(s) to be used for the hybridization in a Coplin jar containing preheated prehybridization buffer and incubate at 42oC for 45 minutes. 4.1.4 Washing Slides - Fill two Coplin jars with MilliQ water and another with isopropanol. - With forceps carefully grasp slide by the labeled end and vertically dip slide into the first Coplin jar (water) so that the slide is completely submerged. Dip slide four or five times. - Dip the slide again in the water five times but only submerging the slide enough to wash the printed array itself. - Using the same technique dip slide into the second Coplin jar (water). - Finally dip slide into the third Coplin jar (isopropanol) submerging the slide completely. Note: Replace each water wash after every five slides. 4.1.5 Drying Slides - Carefully but quickly blow-dry the front of the slide with compressed air. Note: To avoid blowing debris from the forceps onto the array, first blow from where the array ends toward the forceps. Then dry the array itself by blowing down the slide away from the forceps. - Finally dry the back of the slide. - Note the general appearance of the slide. Streaking or mottling on the slide surface indicates further washing is necessary. - Repeat the water/water/isopropanol wash cycle as necessary to clean the slide. Blow-dry between each cycle. 4.1.6 Use slides immediately following prehybridization to ensure optimal hybridization efficiency. 4.2 Hybridization 4.2.1 Prepare 1X hybridization buffer (50% formamide, 5X SSC, and 0.1% SDS). 4.2.2 Prepare Poly(A)-DNA by dissolving stock Poly(A)-DNA in a neutral buffer (i.e. 10 mM Tris, pH 7) to a final concentration of 20 mg/mL. 4.2.3 Prepare COT1-DNA (stock conc.1mg/mL) by ethanol precipitation: - Add 2 to 3 volumes of ethanol and 0.1 volumes of 3 M Sodium Acetate (NaOAc) to the stock tube. - Mix well and place on dry ice for 20-30 minutes or in -20oC freezer overnight. - Centrifuge for 20-30 minutes in a cold room microfuge at maximum angular velocity. - Remove supernatant and allow excess ethanol to dry off. - Dissolve precipitated COT1 in a neutral buffer (i.e. 10 mM Tris, pH 7) to the final concentration of 20mg/mL. 4.2.4 Resuspend labeled probe (Cy3/Cy5 probe mixture: see SOP-M004) in 24 mL of 1X hybridization buffer. Note: Expose Cy labeled probe to light as little as possible during the hybridization process. 4.2.5 To block nonspecific hybridization add: COT1-DNA (20 mg/mL) ... 1mL Poly(A)-DNA (20mg/mL) ... 1mL Note: The COT1-DNA is organism specific: add mouse COT1 to labeled mouse probes and human COT1-DNA to labeled human probes. 4.2.6 To denature, heat the probe mixture at 95oC for 3 minutes and snap cool on ice for 30 sec. 4.2.7 Centrifuge the probe mixture at maximum angular velocity for 1 minute. Keep at room temperature and use immediately. 4.2.8 To Apply Labeled Probe Mixture - Place a prehybridized microarray slide (array side up) between the guide teeth in the bottom half of a hybridization chamber. - Pipette the labeled probe mixture (~26 mL) to the slide surface near one end of the array print area keeping bubbles to a minimum. - Take a 22mm x 60mm microscope glass coverslip, dust it with compressed air, and grasp one end with forceps. - Holding the coverslip over the array print area, lower the end nearest the pool of cDNA probe until solution wicks to the surface of the coverslip. - Gradually lower the opposite end of the coverslip (held by the forceps) onto the slide. The solution may take a minute or two to wick across the entire length of the slide. - After probe has wicked across the slide carefully adjust the coverslips position with the tip of the forceps so that there is an even margin between the edge of the coverslip and the edge of the slide. - Work any large bubbles toward the edge by gently tapping the coverslip surface; small bubbles will absolve themselves during hybridization. 4.2.9 To the small wells at each end of the chamber add 10 mL of water (20mL total), cover, and seal the chamber. 4.2.10 Wrap the chamber in foil (light-tight) and incubate in a 42oC water bath for 16-20 hours. To ensure chamber remains level and does not float to the surface place a small weight upon it. Note: Do not flip the hybridization chamber upside down during hybridization; this may cause the coverslip to shift from the slide and adversely affect the hybridization. 4.2.11 Prepare a low stringency wash buffer (~500mL) containing 1X SSC and 0.2% SDS and a high-stringency wash buffer (~500mL) containing 0.1X SSC and 0.2% SDS. 4.2.12 After the incubation remove foil and unseal hybridization chamber. Remove the slide from the chamber, taking care not to disturb the coverslip. 4.2.13 To remove coverslip submerge slide in a dish containing low stringency wash buffer (preheated to 42oC). With forceps shake the slide gently to loosen the coverslip. With time the coverslip will slide free of the slide surface. Note: Once the slide has been hybridized it should be exposed to light as little as possible. Therefore, all staining dishes should be covered with foil to make them light tight. 4.2.14 After the coverslip is removed place slide in a staining dish containing lower stringency wash buffer (preheated to 42oC) and agitate for 4 minutes. 4.2.15 Wash the slide in a staining dish with high stringency wash buffer by agitating for 4 minutes at room temperature. 4.2.16 Wash the slide in 0.1X SSC agitating for 4 minutes at room temperature. (alternatively one can wash twice in 0.1X SSC agitating for 2.5 minutes to minimize SDS carryover.) 4.2.17 Finally dip the slide in a Coplin jar filled with water several times and blow dry using the same technique as step 4.1.4. 4.2.18 Place slides in a light tight slide box until they can be scanned, preferably as soon as possible.

scan_protocol: Array images were scanned using the GenePix 4000A scanner (Axon Instruments, Union City, CA). Cy5 PM volt at 750, Cy3 PM volt at 680.

data_processing: Array spot intensities were acquired using TIGR Spotfinder as outlined in (Yang et al. 2002, Genome Biol 3: research0062) and normalized using global Lowess regression using MIDAS (Saeed et al. 2003, Biotechniques 34:374-378) set at a smoothing parameter of 0.3 and filtered for inconsistent flip-dye pairs at two standard deviation cutoff.

source_name: C57BL/6J mice on control diet for 21 days

label_protocol: THE INSTITUTE FOR GENOMIC RESEARCH Standard Operating Procedure TITLE: AMINOALLYL LABELING OF RNA FOR MICROARRAYS SOP #: M004 REVISION LEVEL: 2 EFFECTIVE DATE: 3/4/02 AUTHOR: Jeremy Hasseman PRIMARY REVIEWERS: Emily Chen, Ivana Yang 1. PURPOSE This protocol describes the labeling of eukaryotic RNA with aminoallyl labeled nucleotides via first strand cDNA synthesis followed by a coupling of the aminoallyl groups to either Cyanine 3 or 5 (Cy 3/Cy5) fluorescent molecules. 2. SCOPE This procedural format is utilized by Human Colon Cancer and Mouse microarray projects under the supervision of John Quackenbush within the Eukaryotic Genomics Dept. MATERIALS 3.1 5-(3-aminoallyl)-2^deoxyuridine-5^-triphosphate (AA-dUTP) (Sigma; Cat # A0410) 3.2 100 mM dNTP Set PCR grade (Life Technologies; Cat # 10297-018) 3.3 Random Hexamer primers (3mg/mL) (Life Technologies; Cat # 48190- 011) 3.4 SuperScript II RT (200U/uL) (Life Technologies; Cat # 18064-014) 3.5 Cy-3 ester (AmershamPharmacia; Cat # PA23001) 3.6 Cy-5 ester (AmershamPharmacia; Cat # PA25001) 3.7 QIAquick PCR Purification Kit (Qiagen; Cat # 28106) 3.8 RNeasy® Mini Kit (Qiagen; Cat # 74106) 3.9 Microcon YM-30 Centrifugal Filter Devices (Millipore; Cat # 42410) 4. REAGENT PREPARATION 4.1 Phosphate Buffers 4.1.1 Prepare 2 solutions: 1M K2HPO4 and 1M KH2PO4 4.1.2 To make a 1M Phosphate buffer (KPO4, pH 8.5-8.7) combine: 1M K2HPO4 ..9.5 mL 1M KH2PO4 ..0.5 mL 4.1.3 For 100 mL Phosphate wash buffer (5 mM KPO4, pH 8.0, 80% EtOH) mix: 1 M KPO4 pH 8.5... 0.5 mL MilliQ water ... 15.25 mL 95% ethanol ... 84.25 mL Note: Wash buffer will be slightly cloudy. 4.1.4 Phosphate elution buffer is made by diluting 1 M KPO4, pH 8.5 to 4 mM with MilliQ water. 4.2 Aminoallyl dUTP 4.2.1 For a final concentration of 100mM add 19.1 µL of 0.1 M KPO4 buffer (pH 7.5) to a stock vial containing 1 mg of aa-dUTP. Gently vortex to mix and transfer the aa-dUTP solution into a new microfuge tube. Store at -20oC. 4.2.2 Measure the concentration of the aa-dUTP solution by diluting an aliquot 1:5000 in 0.1 M KPO4 (pH 7.5) and measuring the OD289. (Stock concentration in mM = OD289 x 704) 4.3 Labeling Mix (50X) with 2:3 aa-dUTP: dTTP ratio 4.3.1 Mix the following reagents: Final concentration dATP (100 mM) 5µL... (25 mM) dCTP (100 mM) 5µL... (25 mM) dGTP (100 mM) 5µL... (25 mM) dTTP (100 mM) 3µL... (15 mM) aa-dUTP (100 mM) 2µL... (10 mM) Total: 20µL 4.3.1 Store unused solution at -20oC. 4.4 Sodium Carbonate Buffer (Na2CO3): 1M, pH 9.0 4.4.1 Dissolve 10.8 g Na2CO3 in 80 mL of MilliQ water and adjust pH to 9.0 with 12 N HCl; bring volume up to 100 mL with MilliQ water. 4.4.2 To make a 0.1 M solution for the dye coupling reaction dilute 1:10 with water. Note: Carbonate buffer changes composition over time; make it fresh every couple of weeks to a month. 4.5 Cy-dye esters 4.5.1 Cy3-ester and Cy5-ester are provided as a dried product in 5 tubes. Resuspend a tube of dye ester in 73 uL of DMSO before use. 4.5.2 Wrap all reaction tubes with foil and keep covered as much as possible in order to prevent photobleaching of the dyes. Note: Dye esters must either be used immediately or aliquotted and stored at -80oC. Any introduced water to the dye esters will result in a lower coupling efficiency due to the hydrolysis of the dye esters. Since DMSO is hygroscopic (absorbs water from the atmosphere) store it well sealed in desiccant. 5. PROCEDURE 5.1 Aminoallyl Labeling 5.1.1 To 10 ug of total RNA (or 2 ug poly(A+) RNA) which has been DNase I-treated and Qiagen RNeasy purified, add 2 uL Random Hexamer primers (3mg/mL) and bring the final volume up to 18.5 uL with RNase-free water. 5.1.2 Mix well and incubate at 70oC for 10 minutes. 5.1.3 Snap-freeze in dry ice/ethanol bath for 30 seconds, centrifuge briefly at >10,000 rpm and continue at room temperature. 5.1.4 Add: 5X First Strand buffer .. 6 uL 0.1 M DTT .. 3 uL 50X aminoallyl-dNTP mix .. 0.6 uL SuperScript II RT (200U/uL) .. 2 uL 5.1.5 Mix and incubate at 42oC for 3 hours to overnight. 5.1.6 To hydrolyze RNA, add: 1 M NaOH 10 uL 0.5 M EDTA 10 uL mix and incubate at 65oC for 15 minutes. 5.1.7 Add 10 uL of 1 M HCl to neutralize pH. (Alternatively, one can add 25 uL 1 M HEPES pH 7.0 or 25 uL 1 M Tris pH 7.4) 5.2 Reaction Purification I: Removal of unincorporated aa-dUTP and free amines (use either the Qiagen or the Microcon method) Qiagen Cleanup Method: Note: This purification protocol is modified from the Qiagen QIAquick PCR purification kit protocol. The phosphate wash and elution buffers (prepared in 4.1.3 and 4.1.4) are substituted for the Qiagen supplied buffers because the Qiagen buffers contain free amines which compete with the Cy dye coupling reaction. 5.2.1 Mix cDNA reaction with 300 uL (5X reaction volume) buffer PB (Qiagen supplied) and transfer to QIAquick column. 5.2.2 Place the column in a 2 ml collection tube (Qiagen supplied) and centrifuge at ~13,000 rpm for 1 minute. Empty collection tube. 5.2.3 To wash, add 750 uL phosphate wash buffer to the column and centrifuge at ~13,000 rpm for 1 minute. 5.2.4 Empty the collection tube and repeat the wash and centrifugation step (5.2.3). 5.2.5 Empty the collection tube and centrifuge column an additional 1 minute at maximum speed. 5.2.6 Transfer column to a new 1.5 mL microfuge tube and carefully add 30 uL phosphate elution buffer (see 4.1.4) to the center of the column membrane. 5.2.7 Incubate for 1 minute at room temperature. 5.2.8 Elute by centrifugation at ~13,000 rpm for 1 minute. 5.2.9 Elute a second time into the same tube by repeating steps 5.2.6- 5.2.8. The final elution volume should be ~60 uL. 5.2.10 Dry sample in a speed vac. or~ Microcon YM-30 Cleanup Method: 5.2.1 Place the Microcon sample reservoir into a collection microfuge tube. Add 375 uL of water to the cDNA reaction tube and then pipette the sample into the Microcon sample reservoir/collection microfuge tube without touching the membrane. 5.2.2 Centrifuge at 12,000 rpm for 6-10 min. Note: Never centrifuge column to dryness as this will decrease product recovery. Adjust spin time to allow for optimal filtration while allowing enough solution to remain for sufficient recovery. 5.2.3 Empty collection microfuge tube. 5.2.4 To wash add 450 uL of water to the sample reservoir/collection microfuge tube and centrifuge at 12,000 rpm for 6-10 minutes. 5.2.5 Empty collection microfuge tube and repeat previous wash step. 5.2.6 Invert Microcon sample reservoir into a new collection microfuge tube and centrifuge at 12,000 rpm for 1 minute to collect purified sample. 5.2.7 Dry the sample in a speed vac. 5.3 Coupling aa-cDNA to Cy Dye Ester. 5.3.1 Resuspend aminoallyl-labeled cDNA in 4.5 uL 0.1 M sodium carbonate buffer (Na2CO3), pH 9.0. Note: Carbonate buffer changes composition over time so make sure you make it fresh every couple of weeks to a month. 5.3.2 Add 4.5 uL of the appropriate NHS-ester Cy dye (prepared in DMSO: see 4.5) Note: To prevent photobleaching of the Cy dyes wrap all reaction tubes in foil and keep them sequestered from light as much as possible. 5.3.3 Incubate the reaction for 1 hour in the dark at room temperature. 5.4 Reaction Purification II: Removal of uncoupled dye (Qiagen PCR Purification Kit) 5.4.1 To the reaction add 35 uL 100 mM NaOAc pH 5.2. 5.4.2 Add 250 uL (5X reaction volume) Buffer PB (Qiagen supplied). 5.4.3 Place a QIAquick spin column in a 2 mL collection tube (Qiagen supplied), apply the sample to the column, and centrifuge at ~13,000 for 1 minute. Empty collection tube. 5.4.4 To wash, add 0.75 mL Buffer PE (Qiagen supplied) to the column and centrifuge at ~13,000 for 1 minute. Note: Make sure Buffer PE has added ethanol before using (see label for correct volume). 5.4.5 Empty collection tube and centrifuge column for an additional 1 minute at maximum speed. 5.4.6 Place column in a clean 1.5 mL microfuge tube and carefully add 30 uL Buffer EB (Qiagen supplied) to the center of the column membrane. 5.4.7 Incubate for 1 minute at room temperature. 5.4.8 Elute by centrifugation at ~13,000 rpm for 1 minute. 5.4.9 Elute a second time into the same tube by repeating steps 5.4.6- 5.4.8. The final elution volume should be ~60 uL. Note: This protocol is modified from the Qiagen QIAquick Spin Handbook (04/2000, pg. 18). 5.5 Analysis of Labeling Reaction 5.5.1 Use a 50 uL Beckman quartz MicroCuvette to analyze the entire undiluted sample in a spectrophotometer. 5.5.2 Wash the cuvette with water and blow dry with compressed air duster. 5.5.3 Pipette sample into cuvette and place cuvette in spectrophotometer. 5.5.4 For each sample measure absorbance at 260 nm and either 550 nm for Cy3 or 650 nm for Cy5, as appropriate. 5.5.5 Pipette sample from cuvette back into the original sample tube. 5.5.6 For each sample calculate the total picomoles of cDNA synthesized using: pmol nucleotides = [OD260 * volume (uL) * 37 ng/uL * 1000 pg/ng] 324.5 pg/pmol Note: 1 OD260 = 37 ng/uL for cDNA; 324.5 pg/pmol average molecular weight of a dNTP) 5.5.7 For each sample calculate the total picomoles of dye incorporation (Cy3 or Cy5 accordingly) using: pmol Cy3 = OD550 * volume (uL) 0.15 pmol Cy5 = OD650 * volume (uL) 0.25 nucleotides/dye ratio = pmol cDNA pmol Cy dye Note: >200 pmol of dye incorporation per sample and a ratio of less than 50 nucleotides/dye molecules is optimal for hybridizations (see Microarray Cookbook II) 5.5.8 After analysis mix together the two differentially labeled probes (Cy3 vs. Cy5) which will be hybridized to the same microarray slide. 5.5.9 Dry the Cy3/Cy5 probe mixture to completion in a speed vac and continue with SOP: M005 for the hybridization of the probe to a microarray slide.

extract_protocol:
Mice were sacrificed with CO2. Tissues were frozen immediately on dry ice, and stored at –80 ºC until use. Tissues were cut on dry ice and weighed immediately to prevent thawing of tissue.
Trizol Protocol (GibcoBRL): Pooled liver tissues from 4 replicate animals (240mg total) were placed in 6mL Trizol (GibcoBRL), homogenized with polytron homogenizer. Sample was incubated for 5 minutes at room temperature, spun for 10 minutes to remove cellular debris. Chloroform was added to supernatant, shaken by hand for 15 seconds, incubated for 10 minutes at room temperature, and spun for 15 minutes at 12000g at 4ºC. Aqueous phase was transferred to a new tube and 3mL of isopropanol was added, mixed, incubated for 10 minutes at room temperature, and spun for 15 minutes at 12000g at 4ºC. The pellet was washed with 75% ethanol, air dried for 5 minutes, and dissolved in DEPC treated water with heating at 60ºC for 10 minutes. 100ug of RNA were treated with 10 units of DNase (Ambion) in 1mL reaction for 1 hour at 37ºC. RNA were precipitated with 1/10th volume of 3M sodium acetate (pH 5.2) and 2 volumes of absolute ethanol at –80ºC for 30 minutes and spun at 4ºC for 20 minutes. Pellet was dissolved in 100uL of DEPC treated water.
RNeasy Protocol (Qiagen): 350uL of RLT buffer was added to the 100uL of RNA, mixed, then 250uL of absolute ethanol was added. Mixture was placed in RNeasy mini spin-column and centrifuged for 15 seconds at 8000g. The column was washed twice with 500uL of RPE buffer. The column was centrifuged for 15 seconds at 8000g for the first wash and maximum speed for 2 minutes for the second wash. The column was spun one more time at 8000g for 1 minute to remove any remaining buffer. To elute RNA, 30uL of DEPC treated water was added to the column, incubated for 5 minutes, and spun for 1 minute at 8000g. Quality of RNA samples was examined on clean agarose gel and the quantity of RNA was measured with spectrophotometer.

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were continued on the control diet for 21 days, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.

source_name: C57BL/6J mice on folate depletion diet for 14 days followed by 7 days on control diet, both supplemented with 50mM saccharine in drinking water

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days supplemented with 50mM saccharine in drinking water. Mice were then placed back on a control diet (Basal Diet #7024 from TestDiet) for 7 days with 50 mM of saccharine in drinking water. Mice were then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: ApoE knockout (C57BL/6J) mice on folate depletion diet for 14 days followed by 7 days on control diet (BioRep1,TechRep)

description: No additional information

source_name: ApoE knockout (C57BL/6J background) mice on folate depletion diet for 14 days followed by 7 days on control diet

tag: Eight 6-weeks old female B6.129P2-Apoetm1Unc/J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days, then placed back on a control diet (Basal Diet #7024 from TestDiet) for 7 days with the total treatment of 21 days. Mice were then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on folate depletion diet for 14 days supplemented with 50mM saccharine (BioRep1,TechRep)

description: No additional information

source_name: C57BL/6J mice on control diet for 14 days

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were continued on the control diet for 14 days, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days supplemented with 50mM saccharine in drinking water. Mice were then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on folate depletion diet for 14 days with 25mM choline supplement in drinking water (BioRep2,TechRep)

description: No additional information

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days supplemented with 25 mM of choline and 50 mM of saccharine in drinking water. Saccharine was used to reduce the bitter taste of choline. Mice were then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on folate depletion diet for 14 days then 7 days on control, both with 50mM saccharine (BioRep1,TechRep)

description: No additional information


title: C57BL/6J mice on folate depletion diet for 14 days supplemented with 50mM saccharine (BioRep2,TechRep)

description: No additional information


title: C57BL/6J mice on folate depletion diet for 14 days then 7 days on control diet, both with 25mM choline (BioRep1,TechRep)

description: No additional information

source_name: C57BL/6J mice on folate depletion diet for 14 days followed by 7 days on control diet, both supplemented with 25mM choline in drinking water

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days supplemented with 25 mM of choline and 50 mM of saccharine in drinking water. Saccharine was used to reduce the bitter taste of choline. Mice were then placed back on a control diet (Basal Diet #7024 from TestDiet) for 7 days with 25 mM of choline and 50 mM of saccharine in drinking water. Mice were then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: Earthworm exposed to TNT+RDX_Rep2 vs Control_Rep3


title: Earthworm exposed to RDX_Rep1 vs TNT_Rep1

source_name: mRNA from worm (Rep#1) treated with 30 ppm of RDX labeled with Cyanine-3 (green)


title: Earthworm exposed to TNT_Rep2 vs TNT+RDX_Rep3

source_name: mRNA from worm (Rep#2) treated with 50 ppm of TNT labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#3) treated with 50 ppm of TNT and 30 ppm of RDX labeled with Alex Fluor 647 (red)


title: Earthworm exposed to TNT_Rep4 vs TNT+RDX_Rep5

source_name: mRNA from worm (Rep#4) treated with 50 ppm of TNT labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#5) treated with 50 ppm of TNT and 30 ppm of RDX labeled with Alex Fluor 647 (red)


title: Earthworm exposed to Control_Rep4 vs RDX_Rep4

source_name: mRNA from worm (Rep#4) treated with solvent control soil labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#4) treated with 30 ppm of RDX labeled with Alex Fluor 647 (red)


title: ApoE knockout (C57BL/6J) mice on folate depletion diet for 14 days followed by 7 days on control diet (BioRep2,TechRep)

description: No additional information


title: C57BL/6J mice on folate depletion diet for 14 days then 7 days on control diet, both with 25mM choline (BioRep2,TechRep)

description: No additional information


title: ApoE knockout (C57BL/6J background) mice on folate depletion diet for 14 days (BioRep1,TechRep)

description: No additional information

tag: Eight 6-weeks old female B6.129P2-Apoetm1Unc/J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on folate depletion diet for 14 days then 7 days on control diet, both with 50mM saccharine (BioRep2)

description: No additional information

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days supplemented with 50mM saccharine in drinking water.  Mice were then placed back on a control diet (Basal Diet #7024 from TestDiet) for 7 days with 50 mM of saccharine in drinking water. Mice were then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on folate depletion diet for 14 days then 7 days on control diet, both with 50mM saccharine (BioRep1)

description: No additional information


title: C57BL/6J mice on folate depletion diet for 14 days supplemented with 50mM saccharine (BioRep2)

description: No additional information


title: C57BL/6J mice on folate depletion diet for 14 days with 25mM choline supplement in drinking water (BioRep1,TechRep)

description: No additional information


title: C57BL/6J mice on folate depletion diet for 14 days with 25mM choline supplement in drinking water (BioRep1)

description: No additional information


title: ApoE knockout (C57BL/6J background) mice on folate depletion diet for 14 days (BioRep2,TechRep)

description: No additional information


title: C57BL/6J mice on folate depletion diet for 14 days followed by 7 days on control diet, both with 25mM choline (BioRep2)

description: No additional information

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days supplemented with 25 mM of choline and 50 mM of saccharine in drinking water.  Saccharine was used to reduce the bitter taste of choline.  Mice were then placed back on a control diet (Basal Diet #7024 from TestDiet) for 7 days with 25 mM of choline and 50 mM of saccharine in drinking water. Mice were then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on folate depletion diet for 14 days with 25mM choline supplement in drinking water (BioRep2)

description: No additional information

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days supplemented with 25 mM of choline and 50 mM of saccharine in drinking water.  Saccharine was used to reduce the bitter taste of choline.  Mice were then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on folate depletion diet for 14 days supplemented with 50mM saccharine (BioRep1)

description: No additional information

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days supplemented with 50mM saccharine in drinking water.  Mice were then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: ApoE knockout (C57BL/6J background) mice on folate depletion diet for 14 days (BioRep1)

description: No additional information


title: ApoE knockout (C57BL/6J) mice on folate depletion diet for 14 days followed by 7 days on control diet (BioRep1)

description: No additional information


title: C57BL/6J mice on folate depletion diet for 14 days followed by 7 days on control diet, both with 25mM choline (BioRep1)

description: No additional information


title: ApoE knockout (C57BL/6J background) mice on folate depletion diet for 14 days (BioRep2)

description: No additional information


title: ApoE knockout (C57BL/6J) mice on folate depletion diet for 14 days followed by 7 days on control diet (BioRep2)

description: No additional information


title: C57BL/6J mice on folate depletion diet for 14 days followed by 7 days on control diet (21 days total) (TechRep2)

description: No additional information

source_name: Stratagene universal mouse reference RNA

extract_protocol: Total RNA from each cell line were extracted, treated with DNase, and pooled into a single RNA pool. Further details can be obtained from Stratagene.

tag: Stratagene's universal mouse reference RNA (Catalog #740100). Total RNA from 11 mouse cell lines embryo, embryo (fibroblast), kidney, liver (hepatocyte), lung (alveolar macrophasge), B-lymphocyte, T-lymphocyte (thymus), mammary gland, muscle myoblast, skin, testis.

source_name: C57BL/6J mice on folate depletion diet for 14 days followed by 7 days on control diet (21 days total treatment)

extract_protocol:
Mice were sacrificed with CO2. Tissues were frozen immediately on dry ice, and stored at –80 ºC until use. Tissues were cut on dry ice and weighed immediately to prevent thawing of tissue.
Trizol Protocol (GibcoBRL): Pooled liver tissues from 8 replicate animals (240mg total) were placed in 6mL Trizol (GibcoBRL), homogenized with polytron homogenizer. Sample was incubated for 5 minutes at room temperature, spun for 10 minutes to remove cellular debris. Chloroform was added to supernatant, shaken by hand for 15 seconds, incubated for 10 minutes at room temperature, and spun for 15 minutes at 12000g at 4ºC. Aqueous phase was transferred to a new tube and 3mL of isopropanol was added, mixed, incubated for 10 minutes at room temperature, and spun for 15 minutes at 12000g at 4ºC. The pellet was washed with 75% ethanol, air dried for 5 minutes, and dissolved in DEPC treated water with heating at 60ºC for 10 minutes. 100ug of RNA were treated with 10 units of DNase (Ambion) in 1mL reaction for 1 hour at 37ºC. RNA were precipitated with 1/10th volume of 3M sodium acetate (pH 5.2) and 2 volumes of absolute ethanol at –80ºC for 30 minutes and spun at 4ºC for 20 minutes. Pellet was dissolved in 100uL of DEPC treated water.
RNeasy Protocol (Qiagen): 350uL of RLT buffer was added to the 100uL of RNA, mixed, then 250uL of absolute ethanol was added. Mixture was placed in RNeasy mini spin-column and centrifuged for 15 seconds at 8000g. The column was washed twice with 500uL of RPE buffer. The column was centrifuged for 15 seconds at 8000g for the first wash and maximum speed for 2 minutes for the second wash. The column was spun one more time at 8000g for 1 minute to remove any remaining buffer. To elute RNA, 30uL of DEPC treated water was added to the column, incubated for 5 minutes, and spun for 1 minute at 8000g. Quality of RNA samples was examined on clean agarose gel and the quantity of RNA was measured with spectrophotometer.

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days, then placed back on a control diet (Basal Diet #7024 from TestDiet) for 7 days with the total treatment of 21 days.  Mice were then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: A/J mice on folate depletion diet for 14 days followed by 7 days on control diet (21 days total) (TechRep2)

description: No additional information

source_name: A/J mice on folate depletion diet for 14 days followed by 7 days on control diet (21 days total treatment)

tag: Eight 6-weeks old female A/J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days, then placed back on a control diet (Basal Diet #7024 from TestDiet) for 7 days with the total treatment of 21 days.  Mice were then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on folate depletion diet for 14 days followed by 1 day on control diet (15 days total) (TechRep2)

description: No additional information

source_name: C57BL/6J mice on folate depletion diet for 14 days followed by 1 day on control diet (15 days total treatment)

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days, then placed back on a control diet (Basal Diet #7024 from TestDiet) for 1 day with the total treatment of 15 days.  Mice were then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on folate depletion diet for 1 day (TechRep2)

description: No additional information

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 1 day, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: A/J mice on folate depletion diet for 14 days (TechRep2)

description: No additional information

tag: Eight 6-weeks old female A/J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: A/J mice on control diet for 22 days (TechRep2)

description: No additional information

source_name: A/J mice on control diet for 22 days

tag: Eight 6-weeks old female A/J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then continued on control diet for 22 more days, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on folate depletion diet for 2 days (TechRep2)

description: No additional information

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 2 days, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on control diet at time 0 (TechRep2)

description: No additional information

source_name: C57BL/6J mice on control diet at time 0

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were sacrificed on day 0 of experiment (7-weeks old) with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: A/J mice on folate depletion diet for 7 days (TechRep2)

description: No additional information

tag: Eight 6-weeks old female A/J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 7 days, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on control diet for 9 days (TechRep2)

description: No additional information

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were continued on the control diet for 9 days, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: A/J mice on control diet for 9 days (TechRep2)

description: No additional information

source_name: A/J mice on control diet for 9 days

tag: Eight 6-weeks old female A/J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then continued on control diet for 9 more days, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on control diet for 22 days (TechRep2)

description: No additional information

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were continued on the control diet for 22 days, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on folate depletion diet for 14 days (TechRep2)

description: No additional information

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: A/J mice on folate depletion diet for 1 day (TechRep2)

description: No additional information

tag: Eight 6-weeks old female A/J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 1 day, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: A/J mice on folate depletion diet for 14 days followed by 1 day on control diet (15 days total) (TechRep2)

description: No additional information

source_name: A/J mice on folate depletion diet for 14 days followed by 1 day on control diet (15 days total treatment)

tag: Eight 6-weeks old female A/J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days, then placed back on a control diet (Basal Diet #7024 from TestDiet) for 1 day with the total treatment of 15 days.  Mice were then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on folate depletion diet for 7 days (TechRep2)

description: No additional information

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 7 days, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: A/J mice on folate depletion diet for 2 days (TechRep2)

description: No additional information

tag: Stratagene's universal mouse reference RNA (Catalog #740100). Total RNA from 11 mouse cell lines embryo, embryo (fibroblast), kidney, liver (hepatocyte), lung (alveolar macrophasge), B-lymphocyte, T-lymphocyte (thymus), mammary gland, muscle myoblast, skin, testis.

tag: Eight 6-weeks old female A/J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 2 days, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on folate depletion diet for 14 days followed by 1 day on control diet (15 days total) (TechRep1)

description: No additional information


title: C57BL/6J mice on folate depletion diet for 2 days (TechRep1)

description: No additional information


title: C57BL/6J mice on folate depletion diet for 14 days (TechRep1)

description: No additional information


title: C57BL/6J mice on folate depletion diet for 14 days followed by 7 days on control diet (21 days total) (TechRep1)

description: No additional information

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days, then placed back on a control diet (Basal Diet #7024 from TestDiet) for 7 days with the total treatment of 21 days.  Mice were then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on control diet for 9 days (TechRep1)

description: No additional information


title: A/J mice on folate depletion diet for 14 days followed by 7 days on control diet (21 days total treatment) (TechRep1)

description: No additional information

tag: Eight 6-weeks old female A/J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days, then placed back on a control diet (Basal Diet #7024 from TestDiet) for 7 days with the total treatment of 21 days.  Mice were then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on folate depletion diet for 7 days (TechRep1)

description: No additional information


title: Earthworm exposed to TNT_Rep1 vs TNT+RDX_Rep1

source_name: mRNA from worm (Rep#1) treated with 50 ppm of TNT labeled with Cyanine-3 (green)

tag: Adult earthowrms exposed in soil for 28 days.

tag: Worms were snap frozen in liquid nitrogen.

tag: Worm tissue was fixed in RNAlater-ICE (Ambion).

source_name: mRNA from worm (Rep#1) treated with 50 ppm of TNT and 30 ppm of RDX labeled with Alex Fluor 647 (red)

tag: Worm tissue was fixed in RNAlater-ICe (Ambion).


title: C57BL/6J mice on folate depletion diet for 1 day (TechRep1)

description: No additional information

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 1 day, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: C57BL/6J mice on control diet for 22 days (TechRep1)

description: No additional information


title: C57BL/6J mice on control diet at time 0 (TechRep1)

description: No additional information

tag: Eight 6-weeks old female C57BL/6J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were sacrificed on day 0 of experiment (7-weeks old) with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: A/J mice on folate depletion diet for 14 days followed by 1 day on control diet (15 days total treatment) (TechRep1)

description: No additional information


title: Earthworm exposed to RDX_Rep1 vs TNT_Rep2

source_name: mRNA from worm (Rep#1) treated with 30 ppm of RDX labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#2) treated with 50 ppm of TNT labeled with Alex Fluor 647 (red)


title: A/J mice on folate depletion diet for 14 days (TechRep1)

description: No additional information

tag: Eight 6-weeks old female A/J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 14 days, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: Earthworm exposed to RDX_Rep3 vs TNT_Rep3

source_name: mRNA from worm (Rep#3) treated with 30 ppm of RDX labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#3) treated with 50 ppm of TNT labeled with Alex Fluor 647 (red)


title: Earthworm exposed to TNT_Rep5 vs TNT+RDX_Rep5

source_name: mRNA from worm (Rep#5) treated with 50 ppm of TNT labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#5) treated with 50 ppm of TNT and 30 ppm of RDX labeled with Alex Fluor 647 (red)


title: Earthworm exposed to TNT_Rep3 vs TNT+RDX_Rep3

source_name: mRNA from worm (Rep#3) treated with 50 ppm of TNT labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#3) treated with 50 ppm of TNT and 30 ppm of RDX labeled with Alex Fluor 647 (red)


title: Earthworm exposed to RDX_Rep4 vs TNT_Rep4

source_name: mRNA from worm (Rep#4) treated with 30 ppm of RDX labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#4) treated with 50 ppm of TNT labeled with Alex Fluor 647 (red)


title: A/J mice on folate depletion diet for 7 days (TechRep1)

description: No additional information

source_name: Stratagene universal mouse reference RNA

tag: Eight 6-weeks old female A/J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then placed on folate depletion diet (Folate depletion diet #29691 from TestDiet) for 7 days, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: A/J mice on folate depletion diet for 1 day (TechRep1)

description: No additional information


title: Earthworm exposed to RDX_Rep2 vs TNT_Rep2

source_name: mRNA from worm (Rep#2) treated with 30 ppm of RDX labeled with Cyanine-3 (green)


title: Earthworm exposed to TNT_Rep2 vs TNT+RDX_Rep2

source_name: mRNA from worm (Rep#2) treated with 50 ppm of TNT labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#2) treated with 50 ppm of TNT and 30 ppm of RDX labeled with Alex Fluor 647 (red)


title: 24h, GDX+DHT; female

status: Public on Aug 26 2008

city: Quebec

first: Ezequiel

middle: L

last: Calvo

state: Quebec

address: 2705 Boul. Laurier

department: Molecular Endocrinilogy

country: Canada

institute: CRCHUL

description: Gene expression data from adipose tissue, 24h prior to necropsy.

hyb_protocol: Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at 45C to GeneChip MG-U74A v2 Genechip Set. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using an Agilent GeneArray Scanncer (Agilent Technologies).

data_processing: CEL files were extracted with Microarray Suite 5.0 (Affymetrix).

source_name: adipose tissue; 24h, GDX+DHT

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 20 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).

extract_protocol:
Retroperitoneal adipose tissue samples from all mice of the same group were collected, rapidly trimmed, snap-frozen in liquid nitrogen and stored at -80ºC prior to mRNA extraction.
Total RNA was isolated from cells using Trizol Reagent, according to the manufacturer’s protocol (Invitrogen).

treatment_protocol: DHT (0.1mg) was injected subcutaneously 1, 3, 6, 12, 18 and 24 h prior to sacrifice in DHT-treated groups. One group was retained as control and received a vehicle solution.

tag: Female mice retroperitoneal adipose tissue


title: 18h, GDX+DHT; female

description: Gene expression data from adipose tissue, 18h prior to necropsy.

source_name: adipose tissue; 18h, GDX+DHT


title: A/J mice on control diet for 22 days (TechRep1)

description: No additional information

source_name: A/J mice on control diet for 22 days

tag: Eight 6-weeks old female A/J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then continued on the control diet for 22 days, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: A/J mice on control diet for 9 days (TechRep1)

description: No additional information

tag: Stratagene's universal mouse reference RNA (Catalog #740100). Total RNA from 11 mouse cell lines embryo, embryo (fibroblast), kidney, liver (hepatocyte), lung (alveolar macrophasge), B-lymphocyte, T-lymphocyte (thymus), mammary gland, muscle myoblast, skin, testis

source_name: A/J mice on control diet for 9 days

tag: Eight 6-weeks old female A/J mice were purchased from the Jackson laboratory. Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet. Mice were then continued on control diet for 9 more days, then sacrificed with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use. All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle. Mice were housed in micro-ventilated cages with corn cob bedding. Mice were provided with food and water ad libitum.


title: A/J mice on folate depletion diet for 2 days (TechRep1)

description: No additional information


title: Earthworm exposed to TNT_Rep4 vs TNT+RDX_Rep4

source_name: mRNA from worm (Rep#4) treated with 50 ppm of TNT labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#4) treated with 50 ppm of TNT and 30 ppm of RDX labeled with Alex Fluor 647 (red)


title: 6h, GDX+DHT; female

description: Gene expression data from adipose tissue, 6h prior to necropsy.

source_name: adipose tissue; 6h, GDX+DHT


title: 12h, GDX+DHT; female

description: Gene expression data from adipose tissue, 12h prior to necropsy.

source_name: adipose tissue; 12h, GDX+DHT


title: 3h, GDX+DHT; female

description: Gene expression data from adipose tissue, 3h prior to necropsy.

source_name: adipose tissue; 3h, GDX+DHT


title: Earthworm exposed to TNT_Rep3 vs TNT+RDX_Rep4

source_name: mRNA from worm (Rep#3) treated with 50 ppm of TNT labeled with Cyanine-3 (green)


title: 1h, GDX+DHT; female

description: Gene expression data from adipose tissue, 1h prior to necropsy.

source_name: adipose tissue; 1h, GDX+DHT


title: Control, GDX+vehicle; female, replicate

source_name: adipose tissue; Control, GDX+vehicle


title: 6h, GDX+DHT; male

tag: Male mice retroperitoneal adipose tissue


title: Earthworm exposed to RDX_Rep5 vs TNT_Rep5

source_name: mRNA from worm (Rep#5) treated with 30 ppm of RDX labeled with Cyanine-3 (green)

source_name: mRNA from worm (Rep#5) treated with 50 ppm of TNT labeled with Alex Fluor 647 (red)


title: 3h, GDX+DHT; male


title: A/J mice on control diet at time 0 (TechRep1)

description: No additional information

scan_protocol: Array images were scanned using the GenePix 4000A scanner (Axon Instruments, Union City, CA).  Cy5 PM volt at 750, Cy3 PM volt at 680.

data_processing: Array spot intensities were acquired using TIGR Spotfinder as outlined in  (Yang et al. 2002, Genome Biol 3: research0062) and normalized using global Lowess regression using MIDAS (Saeed et al. 2003, Biotechniques 34:374-378) set at a smoothing parameter of 0.3 and filtered for inconsistent flip-dye pairs at two standard deviation cutoff.

source_name: Stratagene universal mouse reference RNA

extract_protocol: Total RNA from each cell line were extracted, treated with DNase, and pooled into a single RNA pool.  Further details can be obtained from Stratagene.

tag: Stratagene's universal mouse reference RNA (Catalog #740100).  Total RNA from 11 mouse cell lines embryo, embryo (fibroblast), kidney, liver (hepatocyte), lung (alveolar macrophasge), B-lymphocyte, T-lymphocyte (thymus), mammary gland, muscle myoblast, skin, testis

source_name: A/J mice on control diet at time 0

extract_protocol:
Mice were sacrificed with CO2.  Tissues were frozen immediately on dry ice, and stored at –80 ºC until use.  Tissues were cut on dry ice and weighed immediately to prevent thawing of tissue.
Trizol Protocol (GibcoBRL): Pooled liver tissues from 8 replicate animals (240mg total) were placed in 6mL Trizol (GibcoBRL), homogenized with polytron homogenizer.  Sample was incubated for 5 minutes at room temperature, spun for 10 minutes to remove cellular debris.  Chloroform was added to supernatant, shaken by hand for 15 seconds, incubated for 10 minutes at room temperature, and spun for 15 minutes at 12000g at 4ºC.  Aqueous phase was transferred to a new tube and 3mL of isopropanol was added, mixed, incubated for 10 minutes at room temperature, and spun for 15 minutes at 12000g at 4ºC.  The pellet was washed with 75% ethanol, air dried for 5 minutes, and dissolved in DEPC treated water with heating at 60ºC for 10 minutes.  100ug of RNA were treated with 10 units of DNase (Ambion) in 1mL reaction for 1 hour at 37ºC.  RNA were precipitated with 1/10th volume of 3M sodium acetate (pH 5.2) and 2 volumes of absolute ethanol at –80ºC for 30 minutes and spun at 4ºC for 20 minutes.  Pellet was dissolved in 100uL of DEPC treated water.
RNeasy Protocol (Qiagen): 350uL of RLT buffer was added to the 100uL of RNA, mixed, then 250uL of absolute ethanol was added.  Mixture was placed in RNeasy mini spin-column and centrifuged for 15 seconds at 8000g.  The column was washed twice with 500uL of RPE buffer.  The column was centrifuged for 15 seconds at 8000g for the first wash and maximum speed for 2 minutes for the second wash.  The column was spun one more time at 8000g for 1 minute to remove any remaining buffer.  To elute RNA, 30uL of DEPC treated water was added to the column, incubated for 5 minutes, and spun for 1 minute at 8000g.  Quality of RNA samples was examined on clean agarose gel and the quantity of RNA was measured with spectrophotometer.

tag: Eight 6-weeks old female A/J mice were purchased from the Jackson laboratory.  Mice were placed on a control diet (Basal Diet #7024 from TestDiet) for one week to become adjusted to this diet.  Mice were sacrificed on day 0 of experiment (7-weeks old) with CO2 and liver tissue was immediately isolated, frozen on dry ice, and stored in -80C until use.  All mice shared the same animal room with controlled temperature (21ºC), humidity, and 12 h light-dark cycle.  Mice were housed in micro-ventilated cages with corn cob bedding.  Mice were provided with food and water ad libitum.


title: 18h, GDX+DHT; male


title: 24h, GDX+DHT; male


title: Control, GDX+vehicle; female


title: Control, GDX+vehicle; male


title: 12h, GDX+DHT; male


title: 1h, GDX+DHT; male


title: Control, GDX+vehicle; male, replicate


title: HFQ_LB_SPI-1_BR2

status: Public on Aug 23 2008

type: mixed

city: Norwich

first: Sacha

last: Lucchini

address: Colney Lane

department: Molecular Microbiology

country: United Kingdom

institute: Institute of Food Research

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

hyb_protocol: Slides blocked with DCE according to protocol at PMID 11266573. For hybridisation and washing protocols see http://www.ifr.ac.uk/safety/Microarrays/default.html#protocols

scan_protocol: Axon GenePix4000A, 10nm resolution scan & see http://www.ifr.ac.uk/safety/Microarrays/default.html#protocols

data_processing: Data centring was performed by bringing the median ratio (Cy5/Cy3) for each of the 16 blocks to one using the following equation: (Ti) = (Cy5i/Cy3i) - c, where T is the centred 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.

organism: Salmonella enterica

source_name: Reference genomic DNA

label_protocol: Channel Label Protocol: Labelling protocol at http://www.ifr.ac.uk/safety/Microarrays/default.html#protocols

growth_protocol: S. Typhimurium grown to stationary phase

extract_protocol: Genomic DNA isolated using the Qiagen 'Genomic DNA' Kit (Cat. No.: 19060 for the buffer kit; 10243 for the columns)

label_protocol: Labelling protocol at http://www.ifr.ac.uk/safety/Microarrays/protocols.html

growth_protocol: S. Typhimurium grown under SPI-1 inducing conditions: overnight growth in LB-broth containing 0.3M NaCl, under oxygen tension.

extract_protocol: Total RNA isolation protocol at http://www.ifr.ac.uk/safety/Microarrays/protocols.html

tag: Salmonella enterica serovar Typhimurium SL1344 hfq-


title: CTRL_LB_SPI-1_BR1

tag: Salmonella enterica serovar Typhimurium SL1344