|
| Status |
Public on Aug 31, 2016 |
| Title |
BPEC_2hr_2Gy_Xrays_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Breast Primary Epithelial Cells, 2Gy, X-ray replicate 1
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Mammary Gland
gender: female
|
| Treatment protocol |
BPECs growing in 25cm2 flasks were irradiated with two (0.02 Gy) and ten automatic shots (0.1 Gy) under a mammography X-ray diagnostic device (SENO DMR plur, General electric). A Therapax STX 150 machine (Pantak) was used to irradiate BPECs at high doses (2Gy). After irradiation, cells were returned to the incubator and maintained for 2 hours until RNA extraction.
|
| Growth protocol |
Breast primary epithelial cells (BPECs) were obtained from normal breast tissue (Instituto Quirúrgico Barcelona) and cultured in WIT medium (Cellaria) using Corning Primaria culture flasks (Corning). Cells growing in 25cm2 flasks were maintained at 37ºC in a humidified incubator with 5% CO2 until irradiation time. Afterwards, cells were returned to the incubator for two hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA collection of the samples was done at three different days in order to have biological triplicates per each sample. Total RNA for microarray analysis was isolated from cells using Trizol reagent (Ambion) and then it was treated with DNase (Qiagen) during RNA purification using RNeasy Kit (Qiagen) following manufacturers instructions. RNA integrity was analized using the Agilent 2100 Bioanalyzer (Agilent)
|
| Label |
Cy3
|
| Label protocol |
cRNA labelling was prepared using 0.5ug of total RNA and using the Low Input Quick Amp Labeling (Agilent) according to the manufacturer's instructions. Labeled/amplified RNA was purified using Qiagen’s RNeasy mini spin columns (Qiagen). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 UV-VIS Spectrophotometer.
|
| |
|
| Hybridization protocol |
First, 0.6ng of labelled cRNA was fragmented at 60°C for 30 minutes following manufacturer’s instructions. Then, Agilent hybridization buffer (Agilent) was added to the fragmentation mixture and hybridized to SurePrint G3 Human Gene Expression v2 Microarray (G4851B, Agilent) for 17 hours at 65°C in a hybridization oven. Microarrays were washed using Gene Expression Wash Buffers (Agilent) following manufacturer’s instructions and recommendations.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using Profile AgilentG3_Gx_1color setting for 8x60K microarrays (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression of breast primary epithelial cells 2 hours after exposure to 2Gy of X-ray under a Therapax device
2GyIR_1
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1_105_Dec08 and Grid: 039494_D_F_20120628) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Dec 28, 2015 |
| Last update date |
Aug 31, 2016 |
| Contact name |
Mariona Terradas |
| E-mail(s) |
mariona.terradas@uab.cat
|
| Organization name |
Universitat Autonoma de Barcelona
|
| Department |
Cell Biology, Animal Physiology and Immunology
|
| Street address |
U. Cell Biology, Fac. Biosciences
|
| City |
Cerdanyola del Valles |
| State/province |
BARCELONA |
| ZIP/Postal code |
08193 |
| Country |
Spain |
| |
|
| Platform ID |
GPL16699 |
| Series (1) |
| GSE76357 |
Coding and Non-coding mRNA expression after exposure of Breast Primary Epithelial Cells to different doses of X-ray |
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