|
| Status |
Public on Jun 05, 2018 |
| Title |
Differentiated stromal viscular cells |
| Sample type |
RNA |
| |
|
| Source name |
Differentiated stromal viscular cells
|
| Organism |
Mus musculus |
| Characteristics |
Sex: male
strain: C57/BL
tissue: Adipose tissue
|
| Treatment protocol |
Epididymal fat from male C57/BL mice were digested with collagenase, and then centrifuged at 9 ×g for 1 sec. The floating cells were separated from the lower phase (SVC). The floating cell fraction was centrifugazed at 226 ×g for 3 min. The resultant floating cells and sediment cells were regarded as mature adipocytes (MA) and SPA, respectively. SVC and SPA were cultured in DMEM. When the cultured cells reached 90% confluency, they were incubated with differentiation medium containing insulin/dexamethasone/IBMX for 3 day followed by subsequent incubation with DMEM for 2 day. The cells were collected and RNA was extracted.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using the Maxwell RSC Simply RNA Tissue Kit (Promega, Fitchburg, WI) following the manufacturer's instructions. The quality of RNA was checked with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The concentration of the RNA solution was determined by NanoView spectrophotometer (General Electric, Boston, MA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 30 ng RNA using the Low Input Quick Amp Labeling Kit, one-color (Agilent) according to the manufacturer's instructions, followed by RNeasy mini kit purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoView spectrophotometer (General Electric).
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome ver.2.0 Microarrays (G2519F #26655) for 17 hours at 65°C in a rotating Agilent hybridization oven. 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), then lightly dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent Microarray Scanner (G2565CA) 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%).
|
| Description |
Gene expression in SVC
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_sep09 and Grid: 026655_D_F_20130207) to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Mar 05, 2018 |
| Last update date |
Jun 05, 2018 |
| Contact name |
Shigeo Takashima |
| E-mail(s) |
staka@gifu-u.ac.jp
|
| Organization name |
Gifu University
|
| Department |
Life Science Research Center
|
| Street address |
1-1 Yanagido
|
| City |
Gifu city |
| State/province |
Gifu |
| ZIP/Postal code |
501-1194 |
| Country |
Japan |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE111399 |
Characteristics of differentiated newly identified adipose precursor cells |
|
