|
| Status |
Public on Sep 11, 2013 |
| Title |
ExVivoBlood_24hr_5Gy_rep4 |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral Blood 24hr
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
age: 46 years
tissue: Peripheral Blood
time: 24hr
treatment: 5Gy
|
| Treatment protocol |
3 ml aliquots of blood were exposed at a rate of 0.82 Gy per minute to 0, 0.5, 2, 5 or 8 Gy gamma-rays using a Gammacell-40 137Cs irradiator, then incubated as above for 48 hours prior to RNA extraction.
|
| Growth protocol |
Freshly drawn peripheral blood from healthy volunteers was irradiated, then diluted 1:1 with RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. The cultures were incubated for 48 hours at 37 ºC in a humidified incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the PerfectPure Blood RNA Purification kit (5 Prime, Inc.) following the manufacturer’s recommendations. The protocol includes differential lysis of red and white blood cells, and an on-column DNase digestion. Globin message was further reduced using GLOBINclear (Ambion Inc., Austin, TX) to specifically remove both alpha- and beta- globin. RNA was quantified using a NanoDrop ND-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 500 ng RNA using the One-Color QuickAmp 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 ml 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 Human Genome Oligo Microarrays (G4112A) 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 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10μm, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded, and only features flagged as Positive and Significant (above background) were included.
|
| |
|
| Submission date |
Feb 08, 2013 |
| Last update date |
Sep 11, 2013 |
| 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 |
GPL6848 |
| Series (1) |
| GSE44201 |
Gene expression in human peripheral blood 48 hours after exposure to ionizing radiation |
|
| Relations |
| Reanalysis of |
GSM226027 |
