|
| Status |
Public on Sep 26, 2014 |
| Title |
PBMC_4h_non-rad rep3 |
| Sample type |
RNA |
| |
|
| Source name |
PBMC 20h rad
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: none
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 |
Sample labeling and hybridization was performed according to the Agilent miRNA Complete Labeling and Hyb Kit (Agilent Technologies) following the manufacturer’s protocol.
|
| |
|
| Hybridization protocol |
Sample labeling and hybridization was performed according to the Agilent miRNA Complete Labeling and Hyb Kit (Agilent Technologies) following the manufacturer’s protocol. Briefly, 100 ng total RNA was labeled and subsequently hybridized overnight (20 hours, 55 °C) to Agilent Human microRNA Microarrays 8x60K v16 using Agilent’s recommended hybridization chamber and oven. Finally, the microarrays were washed once with the Agilent Gene Expression Wash Buffer 1 for 5 min at room temperature followed by a second wash with preheated Agilent Gene Expression Wash Buffer 2 (37 °C) for 5 min.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (Agilent Technologies). The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files. The software determines feature intensities (including background subtraction), rejects outliers and calculates statistical confidences.
|
| Description |
microRNA expression after 20hr 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.
MiRNA data were processed as describe above for mRNA data. However, “Filter on Flags” was used as a filtering step in miRNA samples. Flags are attributes that denote the quality of the entities. These values are generated based on the feature quality on the chip. Genes which are given low significant attribute in the data file would be marked as “Absent” and high significant values would be marked as “Present”.
|
| |
|
| 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 |
GPL16770 |
| Series (2) |
| GSE55954 |
Ionizing Radiation Induced micro RNA 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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