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| Status |
Public on Aug 20, 2015 |
| Title |
Microvesicles derived from 3-month-old Fisher344 rats MSC -3 |
| Sample type |
RNA |
| |
|
| Source name |
3-month-old Fisher344 rats MSC
|
| Organism |
Rattus norvegicus |
| Characteristics |
strain: Fisher344
tissue: Bone Marrow Mesenchymal Stem Cell-derived Microvesicles
age: 3-month-old
|
| Treatment protocol |
Cell debris was removed from the supernatant after centrifugation at 2,000g for 20 min. The supernatant was collected and centrifuged at 100,000g at 4°C for 1 h using a high-speed refrigerated centrifuge (HITACHI CP100WX, Japan).
|
| Growth protocol |
BM-MSCs were extracted from 3-month-old and 24-month-old Fisher344 rats,The isolated primary BM-MSCs were cultured in complete medium (Cyagen Bioscience Inc., USA). After the cells were passaged to P3,Cells were starved overnight in serum-free medium supplemented with 0.5% BSA (Sigma, USA) prior to collection of MVs.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol (Invitrogen) and miRNeasy mini kit (QIAGEN) according to manufacturer’s instructions, which efficiently recovered all RNA species, including miRNAs.
|
| Label |
Hy3
|
| Label protocol |
RNA in 2.0 μL of water was combined with 1.0 μL of CIP buffer and CIP (Exiqon). The mixture was incubated for 30 min at 37°C, and was terminated by incubation for 5 min at 95°C. Then 3.0 μL of labeling buffer, 1.5 μL of fluorescent label (Hy3TM), 2.0 μL of DMSO, 2.0 μL of labeling enzyme were added into the mixture. The labeling reaction was incubated for 1 h at 16°C, and terminated by incubation for 15 min at 65°C.
|
| |
|
| Hybridization protocol |
After stopping the labeling procedure, the Hy3TM-labeled samples were hybridized on the miRCURYTM LNA Array (v.18.0) (Exiqon) according to array manual.
|
| Scan protocol |
Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction.
|
| Data processing |
Replicated miRNAs were averaged and miRNAs that intensities>=30 in all samples were chosen for calculating normalization factor. Expressed data were normalized using the Median normalization.
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| |
|
| Submission date |
Aug 19, 2015 |
| Last update date |
Aug 20, 2015 |
| Contact name |
Yan Wang |
| E-mail(s) |
wangwang_890125@sina.com
|
| Phone |
18262636355
|
| Organization name |
the First Affiliated Hospital of Nanjing Medical University
|
| Street address |
300 Guangzhou Road
|
| City |
Nanjing |
| ZIP/Postal code |
210029 |
| Country |
China |
| |
|
| Platform ID |
GPL19128 |
| Series (1) |
| GSE72198 |
Differentially Expressed microRNAs in Bone Marrow Mesenchymal Stem Cell-derived Microvesicles in Young and Older Rats and Their Effect on Tumor Growth Factor-β1-mediated Epithelial-Mesenchymal Transition in HK2 cells |
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