|
| Status |
Public on May 14, 2015 |
| Title |
High-dose-rate_4Gy_rep5 |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral blood_HDR_4.4Gy_replicate 5
|
| Organism |
Mus musculus |
| Characteristics |
tissue: whole blood
Sex: Male
strain: C57BL/6
treatment: unirradiated
|
| Treatment protocol |
Twenty-four hours after the start of irradiation, mice were euthanized with CO2 and peripheral blood (appx. 0.5 ml) was collected via cardiac puncture, mixed in PAXgene blood RNA solution (1:4 ratio) and stored overnight at 4º C until processing for RNA extraction.
|
| Growth protocol |
Male C57BL/6 mice were exposed with either high-dose-rate (0.85Gy/min) or low-dose-rate (0.0028Gy/min) irradiation to 1.1, 2.2 or 4.4-Gy X-rays. Sham irradiated controls, for both HDR and LDR, were treated exactly the same way as corresponding exposed samples but without irradiation exposure.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The whole blood RNA was extracted using PAXgene blood RNA kit (Qiagen, NV) according to manufacturer’s instruction with on-column DNase I treatment, followed by removal of globin transcripts using the GLOBINclear kit (Ambion Inc., Austin, TX) to remove both a- and b-globin.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using the One-Color Qucik Amp Labeling 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 |
1.65 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x GEx hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112F) 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).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides (Scan setting with eXtended dynamic range, Scan area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to XDR Hi 100% and XDR Lo 10%).
|
| Description |
Gene expression after 24hr in unirradiated mouse blood
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software v10.7 (Agilent) using default parameters to obtain background subtracted and spatially detrended green Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers or Features not Positive and significant were excluded
|
| |
|
| Submission date |
Oct 22, 2014 |
| Last update date |
May 14, 2015 |
| 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 |
GPL10333 |
| Series (1) |
| GSE62623 |
Gene expression in mouse blood following low dose-rate or acute x-ray exposure |
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