|
| Status |
Public on Nov 19, 2019 |
| Title |
Day5 10Gy rep12 |
| Sample type |
RNA |
| |
|
| Source name |
whole blood_Day5 10Gy
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
Sex: male
age: 8-10 weeks
treatment: Day5 10Gy
tissue: whole blood
|
| Treatment protocol |
Mice at 8-10 weeks of age were exposed in the early afternoon to total body radiation doses of 0, 1.5, 3, 6, or 10 Gy using an AECL Gammacell-40 137Cs gamma-ray source at a dose rate 1.05 Gy/ min.
|
| Growth protocol |
Mice were housed under standard vivarium conditions, and all procedures were conducted under an approved IACUC protocol.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were euthanized at 1, 2, 3, 5 or 7 days after irradiation. Blood was obtained via cardiac puncture and immediately added to a tube containing PAXgene Blood RNA stabilization and lysis solution at a ratio of 1:5 (blood:solution) and mixed. RNA was purified following the PAXgene RNA kit recommendations with on-column DNase I treatment, followed by globin reduction using GLOBINclear-Mouse/Rat.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) 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.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarrays 4X44K v2 (G4846A) 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 (G2505B) using one color scan setting for 4x44k v.2 array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 026655_D_F_20100123) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers or not significantly above background were excluded.
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| |
|
| Submission date |
Jan 03, 2019 |
| Last update date |
Nov 19, 2019 |
| Contact name |
Sally Amundson |
| E-mail(s) |
[email protected]
|
| Organization name |
Columbia University
|
| Department |
Center for Radiological Research
|
| Street address |
630 W. 168th St
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE124612 |
Transcriptomic responses in mouse blood during the first week after in vivo gamma irradiation |
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