|
| Status |
Public on Feb 13, 2018 |
| Title |
exosomes_1mM sodium oxalate_48h_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
exosomes
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HK-2
cell compartment: exosomes
|
| Treatment protocol |
The HK-2 cells in the experimental groups were exposed to 1, 2 mM sodium oxalate for 48 h, and the HK-2 cells with PBS was used as control. 200 ml supernatants were centrifuged (Beckman Coulter, USA) at 300×g at 4℃ for 10 min to remove sodium oxalate and detached cells, followed by 2,000×g for 15 min and 10,000×g for 40 min to deplete residual cellar debrisr, and then the supernatant was filtered through a 0.22 µm filter (MerckMillipore, Germany). The filtered supernatant was centrifuged at 110,000×g for 75 min twice by ultracentrifuge (Beckman Coulter, USA). After isolation, exosomes were resuspended in PBS and Stored at -80℃.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol (Invitrogen) and purified with RNeasy mini kit (QIAGEN) according to manufacturer’s instructions. RNA quality and quantity was measured by using nanodrop spectrophotometer (ND-1000, Nanodrop Technologies) and RNA Integrity was determined by gel electrophoresis.
|
| Label |
Cy3
|
| Label protocol |
1μLRNA 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. The Reaction 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.Terminated by incubation for 15 min at 65°C.
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| |
|
| Hybridization protocol |
The total 25 μL mixture from Hy3™-labeled samples with 25 μL hybridization buffer were first denatured for 2 min at 95°C, incubated on ice for 2 min.Then hybridized to the microarray for 16–20 h at 56°C in a 12-Bay Hybridization Systems (Hybridization System - Nimblegen Systems, Inc., Madison, WI, USA).Following hybridization, the slides were achieved, washed several times using Wash buffer kit (Exiqon).
|
| Scan protocol |
The slides were scanned using the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA).Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction.
|
| Description |
sample name: 31
|
| 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. After normalization, significant differentially expressed miRNAs between two groups were identified through Fold change and P-value. Differentially expressed miRNAs between two samples were filtered through Fold change.
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| |
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| Submission date |
Feb 12, 2018 |
| Last update date |
Feb 13, 2018 |
| Contact name |
Xiaofeng Guan |
| E-mail(s) |
18468449@qq.com
|
| Phone |
(+86)18697995523
|
| Organization name |
First Affiliated Hospital of Guangxi Medical University
|
| Street address |
No 6 SHUANGYONG ROAD
|
| City |
Nanning |
| State/province |
Guangxi |
| ZIP/Postal code |
530021 |
| Country |
China |
| |
|
| Platform ID |
GPL17107 |
| Series (1) |
| GSE110509 |
Analysis of microRNA expression profiles in exosomes of proximal renal tubular cells in response to oxalate crystal |
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