|
| Status |
Public on Aug 17, 2015 |
| Title |
Human Mesencyhmal Stem Cells (which exosomes were derived from) sample 2 replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
Human MSC from which exosomes were derived
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human Mesenchymal Stem Cell
rna source: cell line (control)
|
| Growth protocol |
MSCs were expanded in multilayer tissue culture plates (Millipore) in medium depleted of serum derived- microvesicles/exosomes by overnight ultracentrifugation at 100,000 g or in serum reduced mesenchymal stem cell medium (Invitrogen) supplemented with bovine serum albumin (Sigma-Aldrich). Condition medium from 10 x106 MSCs was collected every 24 h and subjected to successive centrifugations at 300 g and 2000 g (10 min. each) to remove cells. The cell-free supernatant was then centrifuged at 10,000 g for 30 min to remove cell debris and 100,000 g (Beckman Coulter Optima L-90K ultracentrifuge) 4°C for 90 minutes. The 10,000 and 100,000 g pellets were washed and re suspended in 100 µl of PBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted and purified using the miRNeasy Mini Kit (217004, Qiagen, Valencia, CA) under fully automated sample preparation by the assistance of the QIAcube device (9001292, Qiagen, Valencia CA) following the manufacturer’s protocol.After extraction, total RNA yield and quality were evaluated using NanoDrop at 260 nm and the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA)
|
| Label |
Cy3
|
| Label protocol |
Labeling reactions were performed for each sample using Agilent miRNA Complete Labeling and Hyb Kit Agilent (5190-0456, Agilent Technologies, Santa Clara, CA). Briefly, with a starting concentration of 100 nanograms of total RNA, an initial cDNA strand was Dephosphorylated using a Phosphatase Treatment, this Dephosphorylated RNA was then reverse transcribed and labeled using Cy3 dye by a reverse transcriptase enzyme. After the cRNA was obtained a purification/desalting step was performed using Micro Bio-Spin P-6 Gel Column Bio-Rad (732-6221, Qiagen, Valencia, CA) followed by a drying of the sample according to the Agilent miRNA Microarray System with miRNA Complete Labeling and Hyb Kit.
|
| |
|
| Hybridization protocol |
The entire sample of Cy3-labelled cRNA was heated at 100°C for 5 minutes in a reaction volume of 10x Agilent blocking agent and 2× Hi-RPM Hybridization Buffer following the manufacturers instructions. On completion of the reaction the mixture was hybridized to Agilent Human miRNA Microarray Kit8x15K V3 (G4470C) for at 55°C for 20 hours in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 5 minutes at room temperature with GE Wash Buffer 1 (Agilent) and 5 minutes with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA High Resolution Microarray Scanner (G2505C US45102918) using one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 3 μm, Tiff 20 bit, Dye channel is set to Green).
|
| Description |
miRNA expression microarrays of exosome from human MSC
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters (miRNA_107_Sep09_patch and Grid: 021827_D_F_20091031 ) to obtain background subtracted and spatially detrended Processed Signal intensities. miRNA expression data was normalized using Genespring 12.6 and performing quantile normalization with no baseline geometric transformation. The file "Normalized_data.txt" contains the normalized signal intensity and is available on the series record.
|
| |
|
| Submission date |
Jul 22, 2015 |
| Last update date |
Aug 17, 2015 |
| Contact name |
Luis A Ortiz |
| E-mail(s) |
lao1@pitt.edu
|
| Phone |
7246121436
|
| Organization name |
University of Pittsburgh
|
| Department |
Environmental and Occupational Health
|
| Lab |
Ortiz Lab
|
| Street address |
100 Technology Drive Room 557
|
| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15219 |
| Country |
USA |
| |
|
| Platform ID |
GPL18743 |
| Series (1) |
| GSE71241 |
Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle micro RNAs |
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