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Sample GSM109253 Query DataSets for GSM109253
Status Public on Sep 01, 2007
Title whole ovary-0ppb TCDD-1
Sample type RNA
Source name whole ovary 0ppb TCDD
Organism Danio rerio
Characteristics Strain: AB
Gender: Female
Age: 6-8 months
Tissue: Whole Ovary
Biomaterial provider Zebrafish International Resource Center, Eugene, OR
Treatment protocol The dietary exposure regimen was designed to expose adult female zebrafish to non-lethal concentrations of TCDD that would yield TCDD egg concentrations ranging from 0 – 13.5 ng TCDD/g egg, which are within the range expected to be toxic to embryos based on the LD50 of 2.5 ng TCDD/g egg (Elonen et al., 1998; Henry et al., 1997). The nominal TCDD concentrations were chosen based on the desired target TCDD concentrations in the eggs following a protocol described by Tietge et al (1998). Tritium-labeled TCDD was synthesized and purified to >99% by the manufacturer (Eagle-Picher, Lenexa, KS, specific activity 47 Ci/mmol). Food yielding a final concentration of 272 ng 3H-TCDD/g food was prepared following a protocol described by Fernandez et al. (1998), with modifications. In brief, the 3H-TCDD stock solution was diluted in acetone, added to trout chow ( Zeigler, Gardner, PA), swirled to ensure homogeneous distribution, and the acetone evaporated from the food in a fume hood overnight. This food was then mixed with uncontaminated trout chow to achieve appropriate concentrations, which were confirmed by liquid scintillation counting to be 0 (acetone only), 10, 40, 100, and 270 ng 3H-TCDD/g food.
Growth protocol Male and female zebrafish were housed separately, and acclimated prior to the initiation of experiments for several weeks at the UWM Marine and Freshwater Biomedical Sciences Center (Milwaukee, WI). Fish were maintained at 26-28ºC on a 14-hour light and 10-hour dark cycle in a flow-through buffered, de-chlorinated water system of the UWM Great Lakes Wisconsin Aquaculture Technology and Environmental Research (WATER) Institute. Fish were spawned once weekly during the experiment. All experimental procedures were approved by the University of Wisconsin-Milwaukee Animal Care and Use Committee.
Extracted molecule total RNA
Extraction protocol Following 15 days of dietary exposure, five females from each treatment group were anesthetized in ice-cold water, and fish were killed by cervical spinal cord transection. Using scissors and forceps cleaned with 0.5M NaOH to reduce RNase contamination, ovaries were removed and immediately frozen in liquid nitrogen and stored at -80C. Total RNA was isolated from individual ovaries homogenized in Trizol reagent (Invitrogen) and further purified using an RNeasy MinElute cleanup kit (Qiagen) as per manufacturers’ protocols. Samples were separated by electrophoresis and stained with ethidium bromide to confirm the integrity of the 18s and 28s ribosomal RNAs, and were quantified by UV spectrophotometry at 260nm using a Nanodrop ND-1000 Spectorphotometer (Nanodrop Technologies, Wilmington, DE).
Label Biotin with streptavidin phycoerythrin
Label protocol First- and second-strand cDNA was synthesized from 15 µg pooled total RNA (3 µg from each of 5 females), and cRNA was synthesized, labeled and fragmented in accordance with standard Affymetrix protocols (Affymetrix, Santa Clara, CA). Following hybridization biotin-labeled cRNAs were stained with phycoerithrin conjugated to streptavidin (Molecular Probes, Eugene, OR).
Hybridization protocol Biotin-labeled cRNAs were hybridized to the zebrafish genome array, washed, and stained with phycoerithrin conjugated to streptavidin (Molecular Probes, Eugene, OR) in accordance with standard Affymetrix protocols (Affymetrix, Santa Clara, CA).
Scan protocol Scanned at 10µM using an Affymetrix scanner. Images were analyzed using Microarray Suite 5.0 (MAS5.0).
Description This study describes the molecular targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) within the zebrafish ovary and insights into TCDD’s ovarian toxicity. More details on the reproductive and physiological endpoints can be found in Toxicological Sciences 87(2) 497-507 (2005) and Toxicological Sciences 90(2) 490-499 (2006).
Data processing MAS5.0
Submission date May 17, 2006
Last update date Apr 30, 2007
Contact name Craig Struble
Phone (414)288-3783
Fax (414)288-5472
Organization name Marquette University
Department Math, Stat, and Comp Sci
Street address P.O. 1881
City Milwaukee
State/province WI
ZIP/Postal code 53201-1881
Country USA
Platform ID GPL1319
Series (1)
GSE4859 Molecular targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) within the zebrafish ovary

Data table header descriptions
VALUE Intensity value determined by MAS5 algorithm
ABS_CALL Present, Absent, or Marginal call determined by MAS5 algorithm
DETECTION P-VALUE P-value associated with ABS_CALL determined by MAS5 algorithm

Data table
AFFX-CreX-5_at 2044.5 P 0.000044
AFFX-CreX-3_at 3108 P 0.000044
AFFX-DapX-5_at 41.2 P 0.000195
AFFX-DapX-M_at 89 P 0.000581
AFFX-DapX-3_at 100.7 P 0.00011
AFFX-LysX-5_at 5.4 A 0.250796
AFFX-LysX-M_at 15.3 A 0.354453
AFFX-LysX-3_at 20.1 P 0.00141
AFFX-PheX-5_at 7.6 A 0.227636
AFFX-PheX-M_at 1.5 A 0.58862
AFFX-PheX-3_at 21.7 A 0.062929
AFFX-ThrX-5_at 9.1 A 0.250796
AFFX-ThrX-M_at 23.6 P 0.009337
AFFX-ThrX-3_at 41.3 P 0.00034
AFFX-TrpnX-5_at 4.5 A 0.455409
AFFX-TrpnX-M_at 0.3 A 0.998407
AFFX-TrpnX-3_at 3.7 A 0.60308
AFFX-r2-Ec-bioB-5_at 46.9 P 0.000244
AFFX-r2-Ec-bioB-M_at 106.3 P 0.000244
AFFX-r2-Ec-bioB-3_at 80.7 P 0.000244

Total number of rows: 15617

Table truncated, full table size 498 Kbytes.

Supplementary file Size Download File type/resource
GSM109253.CEL.gz 3.3 Mb (ftp)(http) CEL
GSM109253.EXP.gz 489 b (ftp)(http) EXP
Raw data provided as supplementary file

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