title: PLO_Field_Soil_rep3

status: Public on Feb 05 2010

city: Amsterdam

first: Benjamin

last: Nota

state: Noord Holland

address: Plesmanlaan 125

department: Research Facilities

country: Netherlands

institute: Sanquin

description: Gene expression after 2 d exposure to PLO field soil

hyb_protocol: 300 ng of Cy3-labeled or Cy5-labeled cRNA (specific activity >8.0 pmol dye/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Custom Gene Expression Microarrays with 8 × 15 k format (GPL7150) for 17 hours at 65°C in a rotating Agilent hybridization oven (10 rpm)  After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), washed in acetonitril for 10 seconds, then dried immediately by air.

scan_protocol: Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using two color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Red&Green and PMT is set to 100%, XDR Hi 100%, XDR Lo 10%).

data_processing: The scanned images were analyzed with Feature Extraction Software 9.5.1.1 (Agilent) using default parameters (protocol GE2-v5_95 and Grid: 016342_D_F_20070323) to obtain mean Red or Green intensities and median Red or Green background intesities. In limma, edwards method for background correction was used (offset = 30), and quantile normalizion was done between all arrays.

organism: Folsomia candida

source_name: PLO, 2d, field soil, replicate 3

label_protocol: Cyanine-3 (Cy3) or Cyanine-5 (Cy5) labeled cRNA was prepared from 500 ng total RNA using the Quick Amp Labeling Kit, two-color (Agilent Technologies) according to the manufacturer’s guidelines with a slight modification where the transcription (labeling) reactions were done in half the volume, followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.

growth_protocol: Cultures were first age synchronized before exposure to soil as follows, 10-20 adult animals were incubated per plastic rings; containing plaster of Paris mixed with charcoal, to lay eggs and were removed after 2 days. The incubation conditions were 20°C with a 12 hour light/dark regime. It took approximately 10 days before the eggs hatched.

extract_protocol: Total RNA was extracted from all animals (~30) per jar, using SV Total RNA isolation System (Promega).

treatment_protocol: Approximately 30 animals, 23 d old, were exposed for two days in glass jars containing ~30 g of: soil spiked with (EC10 or EC50 of) a metal (Ba, Cd, Co, Cr, Pb, or Zn), non-spiked soil, or field soil samples. Exposure was done at 20°C, a 12 h light/dark regime, and 75% humidity. After exposure the animals were extracted from soil with tap water via floatation, put in 1.5 mL tubes and frozen in liquid N2.

tag: sample type

value: pool of ~30 animals

tag: series

tag: classification set

value: field soil set


title: OSRISARRAY9

status: Public on Feb 03 2010

type: RNA

first: Thierry

last: Janssens

address: de Boelelaan 1085

department: Ecological Sciences

institute: VU  University Amsterdam

description: Pool of approximately thirty animals of F.candida

hyb_protocol: Agilent Gene Expression Hybridization Kit (Agilent Technologies) was used for hybridization, which was done at 65°C for 17 hours rotating at 10 rpm in an incubator. After hybridization the arrays were washed using Gene Expression Wash Buffer Kit (Agilent Technologies).

scan_protocol: The arrays were scanned on an Agilent DNA microarray scanner (Agilent Technologies), and fluorescence intensities were measured with Feature Extraction (9.1.3.1) Software (Agilent Technologies).

data_processing: Pre-processing and normalization was done using the limma (2.9.2) package in the R (2.18.3) software environment (http://www.r-project.org/), which consisted of normexp background correction (set off = 30), global loess normalization and between array normalization (Aquantile).

molecule: total RNA

source_name: aniline 77.7 μmole/kg soil (EC50)

label_protocol: 500 ng input was used for amplification and labeling with the Quick Amp Labeling Kit (Agilent Technologies), according to the manufacturer’s guidelines with slightest modification that the transcription (labeling) reactions were done in half amounts of volume. Labeled/amplified cRNA was purified using RNeasy (Qiagen).

treatment_protocol: exposed for 2 days in a jar containing LUFA 2.2 soil spiked with the compound from source

tag: strain

value: Berlin

tag: age

value: 25 days old

source_name: 2,3,4-trichloroaniline 101.8 μmole/kg soil (EC50)


title: C. coli test strain cco054_B

status: Public on Feb 02 2010

type: genomic

city: Ithaca

first: Michael

middle: J

last: Stanhope

state: NY

address: Schurman Hall

department: Population Medicine and Diagnostic Sciences

country: USA

institute: Cornell University

description: CombiMatrix design #5543

hyb_protocol: Standard hybridization conditions for the biotinylated target included preblocking for 30 min at 50°C with 6x SSPE (1x SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.7]) containing 0.05% Tween 20, 5x Denhardt's solution, and 100ng/ul salmon sperm DNA followed by hybridization of the biotinylated target in hybridization solution (6x SSPE, 20-50ng/ul labeled DNA and 0.05% SDS) overnight at 50°C. The arrays were then washed once for at least 15 min with SSPE wash 1 (6x SSPE, 0.05% Tween-20) and then for 1 min with each of the following solutions:  SSPE wash 2 (3x SSPE, 0.05% Tween-20), SSPE wash 3 (0.5x SSPE, 0.05% Tween-20), and PBST wash (2x PBS, 0.1% Tween-20), followed by a final 2x PBS wash at room temperature. The hybridized array was then blocked with 5x casein-PBS buffer (BioFX Laboratories, Owings Mills, MD) for 15 min at room temperature and labeled for 30 min with Cy5-streptavidin (GE Healthcare, Amersham Biosciences, Piscataway, NJ) diluted 1:1,000 in 5x casein-PBS buffer. The arrays were scanned after they were washed twice with each of PBST and PBS solutions.

scan_protocol: Hybridized microarrays covered with the imaging solution and the coverslip were scanned with an Axon GenePix™ 4000B laser scanner (Axon Instruments, Union City, CA) using the following parameters:Wavelengths=635, ScanPower=100, FocusPosition=100, PixelSize=5.

data_processing: After scanning, the image was saved as a .tiff file from which the data were extracted using GenePix software.  The raw hybridization signal values, displayed in the GenePix Pro analysis Results (GPR) file, were log2-transformed to be used in subsequent analysis. Background intensity was estimated as the 75th percentile of all negative control probes, and subtracted on log2 scale from foreground Cy5 intensity of all spots on the array.  Such normalization made the magnitude of signal values of different arrays more comparable, with absent genes on each array centering around 0.  Expectation-maximization (EM) algorithm was then applied on the adjusted probe signals of each array to estimate the means and standard deviations of present genes and absent genes with the following parameters at initiation: mean and standard deviation for genes present and absent are 5.0 and 1.0; 0 and 0.5, respectively, percentage of absent genes at 10%. EM algorithm will run for 100 iterations or stop when the magnitude of change in mean estimate is less than 0.001 between iterations.  The EM algorithm fitted well-established Gaussian mixture model to the normalized signals of each array independently to distinguish the population of absent genes from present genes. It did not rely on comparison to signals of positive and negative control strains, so it was robust to technical processing variation in the array experiment. For each array, genes with spot signal below the lower 0.5 percentile of the estimated distribution of present genes were called absent, i.e. p < 0.005.  For C. coli strains tested on multiple arrays, a gene for a given probe was called present if it had a present call on at least one array.  For genes with multiple probes, a present call ratio, based on the total number of present calls out of the total number of probes for each gene, was used as a measurement of the divergence of the test strain again the reference strain.  The ratio will be in range 0-1, which reflects the gene divergence level (absent to present) in the test strain compared to the reference strain.  The present call ratio 0.4 was chosen as cutoff.  Present genes: ratio >0.4; absent and divergent genes: ratio <=0.4.

label: Biotin

molecule: genomic DNA

organism: Campylobacter coli

source_name: C. coli test strain from bovine

label_protocol: Purified fragments (1 to 2 ug) were labeled with biotin, using Mirus Label IT® µAArray Biotin Labeling Kit  (Mirus Corp., Madison, WI) according to the manufacturer's instructions, followed by removal of unincorporated dyes using QIAquick (Qiagen) columns.

extract_protocol: Genomic DNA was digested by sonication to sizes of 200-400bp, as visualized by agarose gel electrophoresis, then purified by using the Qiagen Qiaquick PCR purification kit.

tag: location

value: USA (WA)


title: E15_2_(3)

status: Public on Dec 01 2009

city: Seattle

first: Ilsa

last: Coleman

state: WA

address: 1100 Fairview Ave N, E2-112

department: Human Biology

institute: Fred Hutchinson Cancer Center

description: Whole male UGS embryonic day 14.5, pool 2 of 3

hyb_protocol: Experimental and reference probes were combined and competitively hybridized to microarrays under a coverslip for 16 h at 63°C.

scan_protocol: Fluorescent array images were collected for Cy3 and Cy5 emissions using a GenePix 4000B fluorescent scanner (Axon Instruments, Foster City, CA). Image intensity data were extracted and analyzed using GenePix 4.1 software.

data_processing: Log2-ratios were normalized using a print-tip specific intensity-based scatter plot smoother using robust locally linear fits to capture the dependence of the log-ratios on overall log-spot intensities

organism: Mus musculus

source_name: E14.5 UGS reference

label_protocol: cDNA probe pairs were prepared by amino-allyl reverse transcription using 2 μg of amplified RNA and labeling with Cy3-dCTP or Cy5-dCTP fluorescent dyes (Amersham Bioscience, Piscataway, NJ.)

extract_protocol: Whole male UGS (E14.5, E15.5, E16.5, E17.5, and E18.5) or separated prostate lobes (P7, P30, and P90) were dissected from C57BL6/J mice and snap frozen in liquid nitrogen. For each biological replication, we pooled 3 to 10 mice representing one or two litters. RNA from pools of UGS or specific prostate lobes (vp, ap, and dlp) was prepared using the Qiagen RNeasy Mini kit. We included an on-column DNaseI treatment to remove contaminating DNA. Before RNA amplification, we combined equal quantities of RNA from vp, ap, and dlp for the postnatal prostate samples. We amplified 1 ug of total RNA from each sample through one round using the Arcturus RiboAmp kit. For the E14.5 UGS reference sample, a second round of amplification was done to provide enough RNA for all microarrays.

tag: gender

value: male

tag: developmental stage

tag: tissue

value: whole UGS pool

tag: arna

value: two round aRNA

value: one round aRNA