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Sample GSM991490 Query DataSets for GSM991490
Status Public on Aug 28, 2012
Title Dioctyl Phathalate 48hr 1000uM GSS164_Overmann_Rat_II_12_75-180
Sample type RNA
 
Source name Primary Rat Hepatocytes
Organism Rattus norvegicus
Characteristics agent: Dioctyl Phthalate
concentration: 1000 uM
time: 48 hours
gender: Male
strain: Sprague Dawley
Treatment protocol On day 3 of culture, cells were dosed with chemical: Fresh 100X chemical dosing solutions in 1% DMSO were prepared 24 h before exposure for all chemical doses. Cells were treated with two concentrations of chemical or vehicle on the morning of day 3 and harvested for RNA at 24 and 48 h post treatment. Prior to RNA isolation all treatments were examined under a light microscope for signs of visible cytotoxicity (i.e. cell death). Five independent cell-cultures (1 well of 6 well plate X 5) per each time point/dose combination were performed to obtain 5 separate biological replicates.
Growth protocol Frozen male Sprague Dawley primary rat hepatocytes were purchased from XenoTech (Lenexa, KS) and stored in the vapor phase of liquid N2, until use. Cells were thawed using supplier’s protocol and XenoTech cell isolation kit (Lenexa, KS). Briefly, one, 4.5 ml cryovial containing 17 x 106 rat hepatocytes was removed from liquid N2 and immediately placed in a shaking 37oC water bath for 2 min. Hepatocytes were transferred to a 50 ml conical tube containing 40 ml of solution A (XenoTech Dulbecco’s Modified Eagles Media supplemented with isotonic Percoll, no pen/strep). The cryovial was rinsed with 4.5 ml of Solution B (XenoTech Dulbecco’s Modified Eagles Media supplemented with isotonic Percoll, no pen/strep) and transferred to the 50 ml conical tube containing cells in solution A. Hepatocytes were centrifuged at 60 g (1000 rpm) using an International Equipment Company Centra-8R Centrifuge (Chattanooga, TN) for 5 min at room temperature. Following centrifugation the supernatant was aspirated and hepatocytes were resuspended in 35 ml of Solution B and centrifuged at 60 g (1000 rpm) for another 3 min at room temperature. The resulting cell pellet was resuspended in 30 ml of XenoTech resuspension media (Lenexa, KS) and cell viability was evaluated by trypan blue exclusion. Following the assessment of viability, cells were resuspended in Modified Chee’s culture media (XenoTech, Lenexa, KS) at a concentration of 0.7 - 1.0 x 106cells/ml, and 2 ml of cell suspension was transferred to each well of 6 well collagen coated plates (Corning Corning, NY) and placed in a 37oC incubator (5% CO2, 80% humidity). Following 3 to 4 hours of incubation to allow hepatocytes to attach, media was removed and 2 ml of ice cold modified Chee’s culture media (XenoTech, Lenexa, KS) supplemented with 0.25 mg/ml BD Matrigel, (BD Biosciences, Bedford, MA) was added to each well using pipets chilled to 4oC. Hepatocytes were incubated at 37oC, 5% CO2, media was replaced every day for the next 2 days with Modified Chee’s culture media (XenoTech, Lenexa, KS).
Extracted molecule total RNA
Extraction protocol Total RNA was isolated at 24 and 48 h post treatment, using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH), further purified using a Qiagen RNeasy kit (Valencia, CA) and RNA integrity was validated using a NanoDrop 8000 Spectrophotometer (Wilmington, DE).
Label biotin
Label protocol Labeled cRNA was synthesized from 1 ug of total RNA from the 24 h and 48 h time point samples using the MessageAmpII Biotin-Enhanced labeling kit (Ambion, Austin, TX) and Agencourt RNAClean beads according to manufacturer’s instructions (Beckman Coulter Inc., Danvers, MA).
 
Hybridization protocol Twenty micrograms of labeled cRNA was fragmented and hybridized to Affymetrix Rat Genome 230 2.0 array (Santa Clara, CA) for 16 h at 45°C. Hybridized arrays were washed using a GeneChip Fluidics Station 450.
Scan protocol GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.
Description Time Course/Dose Response Study
Data processing The data were analyzed with Microarray Suite version 5.0 (MAS 5.0) using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 1500.
 
Submission date Aug 23, 2012
Last update date Aug 28, 2012
Contact name Jay Patrick Tiesman
Organization name Procter & Gamble
Department Global Biotechnology
Lab Mason Business Center
Street address PO BOX 8006
City MASON
State/province OH
ZIP/Postal code 45040-8006
Country USA
 
Platform ID GPL1355
Series (2)
GSE40337 Effect of Dioctyl Phthalate on Rat Primary Hepatocytes.
GSE40348 Hepatotoxicity

Data table header descriptions
ID_REF
VALUE MAS 5.0 signal
ABS_CALL indicating whether the transcript was present (P), absent (A), or marginal (M)
DETECTION P-VALUE

Data table
ID_REF VALUE ABS_CALL DETECTION P-VALUE
AFFX-BioB-5_at 1304.48 P 0.0138105
AFFX-BioB-M_at 1906.89 P 0.000856509
AFFX-BioB-3_at 1491.28 P 0.000126798
AFFX-BioC-5_at 3767.95 P 6.02111e-05
AFFX-BioC-3_at 4571.31 P 0.00010954
AFFX-BioDn-5_at 6275.91 P 0.000445901
AFFX-BioDn-3_at 15835.2 P 8.14279e-05
AFFX-CreX-5_at 43628.4 P 5.16732e-05
AFFX-CreX-3_at 39946.6 P 4.42873e-05
AFFX-DapX-5_at 2364.55 P 4.42873e-05
AFFX-DapX-M_at 6364.22 P 0.000296708
AFFX-DapX-3_at 10616.3 P 4.42873e-05
AFFX-LysX-5_at 588.385 P 0.00010954
AFFX-LysX-M_at 1311.13 P 0.000753643
AFFX-LysX-3_at 3220.49 P 6.02111e-05
AFFX-PheX-5_at 1009.2 P 0.0044838
AFFX-PheX-M_at 1098.18 P 6.02111e-05
AFFX-PheX-3_at 1770.3 P 0.000340305
AFFX-ThrX-5_at 375.387 P 0.000340305
AFFX-ThrX-M_at 876.105 P 0.00359458

Total number of rows: 31099

Table truncated, full table size 938 Kbytes.




Supplementary file Size Download File type/resource
GSM991490_GSS164_Overmann_Rat_II_12_75-180.CEL.gz 2.7 Mb (ftp)(http) CEL
GSM991490_GSS164_Overmann_Rat_II_12_75-180.CHP.gz 165.1 Kb (ftp)(http) CHP
Processed data included within Sample table
Processed data provided as supplementary file

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