|
| Status |
Public on Dec 31, 2013 |
| Title |
(MEFs) by addition of serum from L/MDR-irradiated (20days) female C3H/HeN mice 2 |
| Sample type |
RNA |
| |
|
| Source name |
mouse embryonic fibroblasts
|
| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic fibroblasts
co-culture: serum derived from L/MDR-irradiated female C3H/HeN
irradiation dose: 8Gy
|
| Treatment protocol |
Firstly, Female C3H/HeN mice were irradiated at 4 (10days) or 8 Gy (20days) at L/MDR, and MEFs were cultures with serum derived from these in incubated for 24 hours at 37 ºC in a humidified incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using Trizol (Invitrogene) following the manufacturer's recommendations. The RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.4 ug RNA using the Quickamp lebeling kit (Agilent) according to the manufacturer's instructions (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 40°C for 120 minutes in a reaction volume of 100 ml containing 1x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarrays (G4122F) for 17 hours at 65°C in a rotating Agilent hybridization oven. 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), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner using one color scan setting for 4x44k array slides, (Scan resolution 5 um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after addition of serum derived 8Gy irrdiated mice for 20days
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 11.0 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
May 14, 2012 |
| Last update date |
Dec 31, 2013 |
| Contact name |
Takashi Sugihara |
| E-mail(s) |
sugihara@ies.or.jp
|
| Organization name |
Institute for Environmental Sciences
|
| Department |
Radiobiology
|
| Street address |
2-121 Hacchazawa
|
| City |
Rokkasho |
| State/province |
Aomori |
| ZIP/Postal code |
039-3213 |
| Country |
Japan |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE37958 |
Microarray analysis of gene expression in mouse embryonic fibroblasts (MEFs) by addition of serum from L/MDR irradiated mice at 10 and 20 days. |
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