|
| Status |
Public on Jun 30, 2018 |
| Title |
ATSC_IRI_rep01 |
| Sample type |
RNA |
| |
|
| Source name |
Adipose tissue deirved mesenchymal stem cells, ischemia-reperfusion injury, replicate 1
|
| Organism |
Rattus norvegicus |
| Characteristics |
tissue: Adipose tissue
cell type: mesenchymal stem cells
|
| Treatment protocol |
ATSC were harvested from the subcutaneous fat of male Sprague-Dawley rats. The subcutaneous adipose tissue was washed \with sterile phosphate buffered saline (PBS) and then treated with 0.1% collagenase in PBS for 30 minutes at 37°C with gentle agitation. After filtration through 100-μm mesh filter to remove debris, the filtrate was washed three times and completely suspended in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine. The cultures were maintained in an incubator with a humidified atmosphere of 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction was performed using TRIzol® Reagent (Invitrogen, Carlsbad, CA) and the extraction was done according to the manufacturer’s instructions. The quantity and quality of each RNA sample was determined using NanoDrop ND-2000 spectrophotometer (Thermo Scientific, Wilmington, DE) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) RNA Pico Kit.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using Agilent One-Color Quick Amp labeling kit 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-2000 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 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent Hi-RPM hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Rat Genome Microarray (G4131F) for 17 hours at 65°C in a rotating Agilent hybridization oven at 10 rpm. 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 (G2505C) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Jun 17, 2013 |
| Last update date |
Jun 30, 2018 |
| Contact name |
Kwong Wai CHOY |
| Organization name |
CUHK
|
| Department |
O and G
|
| Street address |
Hong Kong
|
| City |
Hong Kong |
| ZIP/Postal code |
852 |
| Country |
Hong Kong |
| |
|
| Platform ID |
GPL7294 |
| Series (1) |
| GSE48011 |
Mesenchymal stem cells gene expression on liver after ischemia-reperfusion injury. |
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