|
| Status |
Public on Feb 08, 2011 |
| Title |
Astrocyte_miR-29-transfected_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
mus musculus fetal astrocyte, miR-29-transfected
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
age: embryonic day 18
cell type: astrocyte, primary culture
|
| Biomaterial provider |
Kinki University, Japan
|
| Treatment protocol |
Astrocytes were transfected in six-well plates (AGC Techno Glass, Japan) containing the medium supplemented with 80 nM each of miR-29 (Ambion Pre-miR hsa-miR-29a/b/c Precursors) or control miRNA (Ambion Pre-miR Negative Control1) using RNAiMax (Invitrogen) in accordance with manufacturer’s protocols. miR-29 RNA for the transfection was prepared by mixing the three miR-29 isoforms in a 1:2:1 molar ratio of Pre-miR-29a:-29b:-29c Precursors. The transfection was conducted three weeks after the initial plating. 48 hr posttransfection, cells were harvested and processed for preparation of RNA samples. The transfection efficiency was above 90% as determined using a fluorochrome-conjugated miR-29 precursor (TAKARA BIO, Japan). RNA extraction was performed in triplicate for each cell type. Total RNA was isolated from the transfected astrocyte cells using an RNAiso Plus (TAKARA BIO). The quality of RNA was assessed using an Agilent 2100 Bioanalyzer, which is indicative of integrity of every RNA sample sufficient for downstream assays.
|
| Growth protocol |
Primary astrocyte cultures prepared from cerebral cortex of fetal C57BL/6 mouse (embryonic 18-day old) were purchased from ScienCell Research Laboratories. Cells were always cultured and maintained in poly-L-lysine-coated plastic wares (AGC) supplied with Astrocyte Medium (ScienCell) following the supplyer’s recommendations.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from the transfected astrocyte cells using an RNAiso Plus (TAKARA BIO).
|
| Label |
Cy3
|
| Label protocol |
The labeling reaction was carried out separately for the RNA samples using a Quick Amp Labeling Kit one-color, in the presence of cyanine 3-CTP.
|
| |
|
| Hybridization protocol |
The dye-labeled target (1.6 μg as cRNA) was fragmented and hybridized on an Agilent Whole Mouse Genome 4X44K microarray at 65°C for 17 hr with a Gene Expression Hybridization Kit. The hybridized slide was washed in Gene Expression Wash Buffer 1 at room temperature for 1 min, which was followed by a wash for 1 min in Gene Expression Wash Buffer 2.
|
| Scan protocol |
The processed microarrays were scanned using an Agilent DNA Microarray Scanner.
|
| Data processing |
Data extraction from raw image files was done with Agilent Feature Extraction software ver.10.7.
|
| |
|
| Submission date |
Feb 02, 2011 |
| Last update date |
Feb 08, 2011 |
| Contact name |
Hiroyuki Tanabe |
| Organization name |
Kinki University
|
| Department |
Faculty of Agriculture Department of Advanced Bioscience
|
| Street address |
3327-204 Nakamachi
|
| City |
Nara City |
| State/province |
Nara |
| ZIP/Postal code |
631-8505 |
| Country |
Japan |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE27035 |
Global profiling of gene expression in mouse astrocyte in response to the potential longevity determinant miR-29 |
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