|
| Status |
Public on Apr 29, 2014 |
| Title |
BMSCs from age mice 3 |
| Sample type |
RNA |
| |
|
| Source name |
aged mice 3
|
| Organism |
Mus musculus |
| Characteristics |
background strain: C57BL/6
tissue: bone marrow
gender: female
age: 18 months
|
| Treatment protocol |
1ml Trizol was added to BMSCs per 3.5cm dish and then transferred into freezing tubes.The tubes were preserved in liquid nitrogen and then sent to company in dry ice.
|
| Growth protocol |
We collected bone marrow cells from 3 or 18 months old female C57BL/6 mice and then incubated the cells aliquots for 20 min at 4°C with phycoerythrin (PE)-, FITC-, peridinin chlorophyll protein (Per CP)- and allophycocyanin (APC)-conjugated antibodies to mouse Sca-1, CD29, CD45 and CD11b. Acquisition was performed on a fluorescence-activated cell sorting (FACS) Aria model, and the analysis was performed using FACS DIVE software.The sorted CD29+Sca-1+CD45−CD11b− cells were BMSCs.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was extracted by mirVanaTM RNA Isolation Kit (Applied Biosystem p/n AM1556 ) following the manufacturer's instructions
|
| Label |
cy3
|
| Label protocol |
0.1ug total RNA were dephosphorylated, denaturated and then labeled with Cyanine-3-CTP（Cy3）using the Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions,then followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 UV-VIS Spectrophotometer version 3.2.1.
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| |
|
| Hybridization protocol |
0.6 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 25ul containing 25x Agilent fragmentation buffer and 10x Agient blocking agent following the manufacturers' instructions. On completion of the fragmentation reaction, 25ul of 2x GEx Hybridization Buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent-046065 Mouse miRNA Microarray V19.0 8x60K(Agilent) 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 dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
microRNAs expression in aged mice
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities as the raw data. Raw data were normalized in quantile algorithm with Genespring 12.0(Agilent). Probe that at least 100.0 percent of samples in any 1 condition out of 2 conditions have flags in Detected were maintained.
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| |
|
| Submission date |
Apr 28, 2014 |
| Last update date |
Apr 29, 2014 |
| Contact name |
Peng Cheng |
| E-mail(s) |
[email protected]
|
| Phone |
8613160090106
|
| Organization name |
Central South University
|
| Department |
The Second Xiangya Hospital
|
| Lab |
Institute of Endocrinology & Metabolism
|
| Street address |
139# Middle Renmin Road
|
| City |
Changsha |
| State/province |
Hunan |
| ZIP/Postal code |
410011 |
| Country |
China |
| |
|
| Platform ID |
GPL17912 |
| Series (1) |
| GSE57127 |
MicroRNA-188 regulates age-related switch between osteoblast and adipocyte differentiation |
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