|
| Status |
Public on Sep 26, 2014 |
| Title |
PBMC_4h_rad rep3 |
| Sample type |
RNA |
| |
|
| Source name |
PBMC 4h rad
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: ionizing radiation
time: 4h
cell type: peripheral blood mononuclear cells
donor: 3
|
| Treatment protocol |
PBMC were obtained from 4 healty volunteers by venous blood draw. Cells were separated by Ficoll-Paque density gradient centrifugation. In short, heparinized anticoagulated blood specimens were processed immediately after venipuncture, diluted 1:2 in Hanks balanced salt solution and shifted carefully into 50 milliliter (ml) tubes containing Ficoll‐Paque solution. Tubes were centrifuged for 15 minutes at 800g at room temperaturewithout brake and buffy coats with mononuclear cells were obtained. Cells werewashed in HBSS and resuspended CellGro serum-free medium. Cells were irradiated with Caesium-13760 Gray.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At the end of incubation time (2h, 4h, and 20 h) as well as immediately after PBMC separation (0h – only 1 sample per donor) total RNA was isolated of irradiated and non-irradiated PBMCs, by using Trizol® Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer´s instructions. Total RNA quantification was performed using a NanoDrop-1000 spectrophotometer (Peglab, Erlangen, Germany) and RNA quality was monitored with Agilent 2100 Bioanalyzer (Agilent, Böblingen, Germany).
|
| Label |
Cy3
|
| Label protocol |
For the linear T7-based amplification step, 100 ng of each total RNA sample was used. To produce Cy3-labeled cRNA, the RNA samples were amplified and labeled using the Agilent Low Input Quick Amp Labeling Kit (Agilent Technologies) following the manufacturer’s protocol. Yields of cRNA and the dye-incorporation rate were measured with the ND-1000 Spectrophotometer (NanoDrop Technologies).
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| |
|
| Hybridization protocol |
The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, 600 ng Cy3-labeled fragmented cRNA in hybridization buffer was hybridized overnight (17 hours, 65 °C) to Agilent W hole Human Genome Oligo Microarrays 8x60K using Agilent’s recommended hybridization chamber and oven.Finally, the microarrays were washed once with the Agilent Gene Expression Wash Buffer 1 for 1 min at room temperature followed by a second wash with preheated Agilent Gene Expression Wash Buffer 2 (37 °C) for 1 min. The las t washing step was performed with acetonitrile.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (Agilent Technologies). All Cy3 images can be found on the provided CD-ROM.
|
| Description |
Gene expression after 4hr in irradiated PBMC
|
| Data processing |
The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files. The software determines feature intensities (including ackground subtraction), rejects outliers and calculates statistical confidences. Background-corrected fluorescence intensity values were imported into GeneSpring v.11 log2-transformed and then normalized by quantile-normalization. GeneSpring calculates the mean values of identical replicate probes on each chip.
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| |
|
| Submission date |
Mar 17, 2014 |
| Last update date |
Sep 26, 2014 |
| Contact name |
Lucian Beer |
| Organization name |
Medical University of Vienna
|
| Department |
Department of Surgery
|
| Lab |
Christian Doppler Laboratory for the. Diagnosis & Regeneration of Cardiac and Thoracic Diseases
|
| Street address |
Währginer Gürtel 18-20
|
| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
| |
|
| Platform ID |
GPL14550 |
| Series (2) |
| GSE55953 |
Ionizing Radiation Induced Gene Expression Alterations in Human Peripheral Blood Mononuclear Cells |
| GSE55955 |
Ionizing Radiation Induced Gene and micro RNA Expression Alterations in Human Peripheral Blood Mononuclear Cells |
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