|
| Status |
Public on Jan 31, 2011 |
| Title |
TBI Blood_4hr_1.25Gy_rep1_091 |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral Blood, 4hr, 1.25Gy, replicate 1
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: whole blood
gender: male
age: 53 years
disease status: TBI patient #091
treatment group: 4hr, 1.25Gy
|
| Treatment protocol |
Peripheral blood was collected several hours before the initial fraction of total body irradiation (TBI), then at 4 hr after first 1.25-Gy fraction and at 1 day after the first fraction. The 1 day samples included exposure to three 1.25-Gy fractions with approximately 4 hr between fractions and received a total exposure of 3.75-Gy. Blood was drawn into PAXgene blood RNA tubes (PreAnalytiX GmbH, USA) and kept at room temperature at least 6 hrs before RNA isolation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the PAXgene Blood RNA Kit (Qiagen/BD company, TX) according to manufacturer’s instructions, followed by removal of globin transcripts using the GLOBINclear kit (Ambion Inc., Austin, TX) to remove both a- and b-globin. 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 0.5 ug 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 (specific activity >10.0 pmol Cy3/ug 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 4hr with 1.25Gy-TBI cancer patient blood
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1.3.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 014850_D_20070207) 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.
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| |
|
| Submission date |
Feb 02, 2010 |
| Last update date |
Jan 31, 2011 |
| Contact name |
Sally Amundson |
| E-mail(s) |
saa2108@cumc.columbia.edu
|
| 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 |
GPL6480 |
| Series (1) |
| GSE20162 |
Prediction of in vivo radiation dose in radiotherapy patients using ex vivo and in vivo gene expression signatures |
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