|
| Status |
Public on Feb 22, 2017 |
| Title |
blood 1Gy Neutron rep6 |
| Sample type |
RNA |
| |
|
| Source name |
blood 1Gy Neutron
|
| Organism |
Homo sapiens |
| Characteristics |
donor: Donor 6
tissue: peripheral blood
|
| Treatment protocol |
Peripheral blood from healthy volunteers was drawn into 0.105 mol/l sodium citrate Vacutainer tubes (Becton Dickinson and Company, Franklin Lakes, NJ). The blood was divided into 3-ml aliquots and exposed to either X-rays or IND-spectrum neutrons (PMID 26414507).
|
| Growth protocol |
After irradiation, blood samples were diluted 1:1 with RPMI 1640 medium (Mediatech, Herndon, VA) supplemented with 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT), as previously described (Grace et al., Int J Radiat Biol 2002; 78:1011-21; PMID 12456288), and were incubated for 24 h at 37º C in a humidified incubator with 5% CO2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The RNA was prepared using the PerfectPure RNA blood kit (PerfectPure, Gaithersburg, MD) following the manufacturer’s recommendations. This protocol differentially lyses red and white blood cells in whole blood to preferentially deplete globin mRNA. Remaining globin mRNA was further reduced using GLOBINclear (Ambion Inc., Austin, TX) specifically to remove both a- and b-globin. 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 100 ng RNA using the One-Color Low Input Quick 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%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7 (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. Data were filtered to exclude features not positive and significant in ≥25% of the samples, or those without at least a 1.5-fold change in expression values across the samples.
|
| |
|
| Submission date |
Dec 05, 2016 |
| Last update date |
Feb 24, 2017 |
| 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 |
GPL13497 |
| Series (1) |
| GSE90909 |
Gene expression response in human peripheral blood exposed to x-rays or neutrons ex vivo |
|
