|
| Status |
Public on Jun 24, 2013 |
| Title |
TCD4_2H30_D001Gy_Ind4 |
| Sample type |
RNA |
| |
|
| Source name |
Lymphocyte TCD4, 150min, 10mGy, Individual4
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Lymphocyte TCD4
gender: male
|
| Treatment protocol |
Human whole blood samples from 5 healthy donors were diluted in IMDM medium (1:10) and exposed to 500, 100, 50, 25, 10 and 5 mGy of Cobalt 60 gamma-rays. Gene expression alterations were measured 150, 300, 450 and 600 min post exposure after negative cell sorting of lymphocytes TCD4+ cells.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from lymphocyte T CD4+ using RNeasy Mini columns from the miRNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug total 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). 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 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4845A) 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 (G2565AA) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um double pass, 16 bit Dye channel is set to Green and Green PMT is set to 100%, XDR set to 0.1).
|
| Description |
Gene expression in Lymphocyte TCD4 150min post-exposure to 10mGy of human whole blood:IMDM (1:10)
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 7.5 (Agilent) using default parameters (protocol GE1-107_sep08 and Grid: 026652_D_F_20101031) to obtain raw signal intensities. Microarray raw data were analyzed, normalized and filtered with R software, limma and Agi4x44PreProcess package of the Bioconductor repository. Briefly, the mean signal intensity for each spot was used and a log base 2 transformation was performed. The normalization step was performed using the quantile method of the normalizeBetweenArrays function of the limma package. Agilent Feature Extraction Software flagged undetected spot and bad features allowing excluding them from the analysis. The filtering criteria consisted of removing a probe when it was undetected in at least 75% of the replicates. A total of 19,246 probes were selected after filtering step.
|
| |
|
| Submission date |
Dec 26, 2012 |
| Last update date |
Jun 24, 2013 |
| Contact name |
Gaetan GRUEL |
| E-mail(s) |
gaetan.gruel@irsn.fr
|
| Organization name |
Institut de Radioprotection et de Surete Nucleaire
|
| Department |
Service de RadioBiologie et d'Epidemiologie
|
| Lab |
Laboratoire de Dosimetrie Biologique
|
| Street address |
BP 17
|
| City |
Fontenay aux Roses Cedex |
| ZIP/Postal code |
92262 |
| Country |
France |
| |
|
| Platform ID |
GPL13497 |
| Series (1) |
| GSE43151 |
Characterization of gene expression profiles at low and very low doses of ionizing radiation |
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