|
| Status |
Public on Aug 21, 2010 |
| Title |
Methoxychlor (50uM) rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
Neonatal mouse ovaries cultured in Methoxychlor (50uM)
|
| Organism |
Mus musculus |
| Characteristics |
strain: Swiss
tissue: Ovary
agent: Methoxychlor (50uM)
replicate: 2
age: Post natal day 3-11
|
| Treatment protocol |
Ovaries were treated with vehicle control medium (0.1% DMSO), VCD (50µM), MXC (50µM), or MEN (5µM). Xenobiotic culture concentrations were determined by pilot studies performed in our laboratory with the intention of inducing overt toxicity.
|
| Growth protocol |
Swiss neonates were sacrificed by CO2 inhalation followed by decapitation. Ovaries were excised, trimmed of excess tissue and placed on culture plate inserts in 6-well tissue culture plate wells floating atop 1.5ml DMEM/F12 medium containing 5% (v/v) fetal calf serum, 1mg/ml bovine serum albumin, 50µg/ml ascorbic acid, 27.5μg/ml insulin–transferrin–selenium, 2.5 mM glutamine and 5U/ml penicillin/streptomycin. Media were supplemented with 40 ng/ml basic fibroblast growth factor, 50 ng/ml leukemia inhibitory factor, and 25 ng/ml stem cell factor. Using fine forceps a drop of medium was placed over the top of each ovary to prevent drying. Ovaries were cultured for 4 days at 37°C and 5% CO2 in air, with media changes every two days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from ovaries using two rounds of a modified acid guanidinium thiocyanate–phenol–chloroform protocol: washed cells resuspended in lysis buffer (4 M guanidinium thiocyanate, 25 mM sodium citrate, 0.5% sarkosyl, 0.72% β-mercaptoethanol). RNA was isolated by phenol/chloroform extraction and isopropanol precipitated.
|
| Label |
biotin
|
| Label protocol |
Mouse total RNA was quality ascertained using the Agilent Bioanalyser 2100 using the NanoChip protocol. A total of 7ug was labelled using the Affymetrix One Cycle cDNA synthesis kit (Millenium Sciences cat no. 900431). The subsequent cDNA was cleaned using the Affymetrix GeneChip Sample Cleanup kit (Millenium Sciences cat no. 900371). Upon cleaning of the cDNA the next step was to incorporate biotin into the resultant cRNA using the Affymetrix IVT labelling kit (Millenium Sciences cat no. 900449). After the IVT process the labelled cRNA was cleaned using the above GeneChip Sample Cleanup kit. The quantity of product was ascertained using the Agilent Bioanalyser 2100 using the NanoChip protocol. A total of 20ug of labelled cRNA was then fragmented to the 50-200bp size range and quality control checked using the Bioanalyser 2100 using the NanoChip protocol.
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| |
|
| Hybridization protocol |
Samples that pass QC check are then prepared for hybridisation to the Mouse 430 version 2.0 GeneChip by preparing a probe cocktail (cRNA @ 0.05ug/ul) that includes 1x Hybridisation Buffer (100mM MES, 1m NaCl, 20mM EDTA, 0.01% Tween-20), 0.1mg/ml Herring Sperm DNA, 0.5mg/ml BSA, and 7% DMSO. A total hybridisation volume of 300ul is prepared for each sample and 200ul loaded into a Mouse 430 version 2.0 GeneChip. The chip is hybridised at 45C for 16 hours in an oven with a rotating wheel at 60rpm. After hybridisation the chip is washed using the appropriate fluidics script in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
Upon completion of the washing, the chips are then scanned using the Affymetrix GeneChip Scanner 3000. The scanner operating software, GCOS, converts the signal on the chip into a DAT file, which will then be used for generating subsequent CEL and CHP files for analysis.
|
| Data processing |
The data were analyzed with Partek software (Partek Genomics Suite) and were normalised using the Robust Multichip Average method using the DMSO treatment as a control.
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| |
|
| Submission date |
Aug 20, 2010 |
| Last update date |
Aug 20, 2010 |
| Contact name |
Alexander Peter Sobinoff |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Newcastle Australia
|
| Department |
Science and IT
|
| Lab |
Reproductive Science Group
|
| Street address |
University Drive
|
| City |
Newcastle |
| State/province |
NSW |
| ZIP/Postal code |
2308 |
| Country |
Australia |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE23725 |
Expression data from mouse neonatal ovarian xenobiotic culture experiments |
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