|
| Status |
Public on Feb 12, 2019 |
| Title |
Exo-CPCcomm _20171005_Lei ON 244643_1_12 |
| Sample type |
RNA |
| |
|
| Source name |
commerical cardiac progenitor cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: exosomes
|
| Treatment protocol |
Human iPSC cell line ACS-1021 (ATCC, USA), and CPCs induced by ISX-9 were cultured. In some cases, EB and commercial human CPCs were also cultured.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Conditioned medium was collected and exosomes were isolated by centrifugation at 3000 rpm for 30 min to remove cells and debris, followed by filtration through a 0.22 μm filter to remove the remaining debris. Then the medium was further concentrated to 500 μl using Amicon Ultra-15 100 kDa centrifugal filter units (Millipore). Isolation of exosomes in the concentrated medium was carried out through qEV size exclusion columns (Izon Science). Exosome fractions were collected and concentrated by Amicon Ultra-4 10 KDa centrifugal filter units to a final volume of <100 μl.
|
| Label |
multiplexed DNA tags (miR-tag)
|
| Label protocol |
The assay allows measurement of 800 different microRNAs at the same time in each sample. 3.5 μl of suspension RNA was annealed with multiplexed DNA tags (miR-tag) and bridges target specifics. Mature microRNAs were bonded to specific miR-tags using a Ligase enzyme, and excess tags were removed by enzyme clean-up step.
|
| |
|
| Hybridization protocol |
The tagged microRNA product was diluted 1 to 5, and 5 μl was combined with 20 μl of Reporter probes in hybridization buffer and 5 μl of Capture probes overnight (17 hours) at 65°C to permit hybridization of probes with specific target sequences. Excess probes were removed using two-step magnetic bead-based purification on an automated fluidic handling system (nCounter Prep Station) and target/probe complexes were immobilized on the cartridge for data collection.
|
| Scan protocol |
The nCounter Digital Analyzer took images of immobilized fluorescent reporters in the sample cartridge with a CCD camera through a microscope objective lens. For each cartridge, a high-density scan encompassing 325 fields of view was performed.
|
| Description |
Commercial human CPCs derived from human iPS cells (Catalog: R1093, Cellular Dynamics International)
|
| Data processing |
NanoString raw data was analyzed with nSolver™ software, provided by NanoString Technologies. The mean plus 2 times the standard deviation of Negative Control Probes was used to perform background subtraction; positives were used to perform technical normalization to adjust lane by lane variability due to differences in hybridization, purification or binding.
Data was then normalized by calculating the geometric mean of the spikes present in each sample, as recommended by NanoString.
|
| |
|
| Submission date |
Feb 11, 2019 |
| Last update date |
Feb 12, 2019 |
| Contact name |
Wanling Xuan |
| E-mail(s) |
wxuan@augusta.edu
|
| Phone |
6184131805
|
| Organization name |
Augusta University
|
| Department |
Vascular Biology Center
|
| Street address |
1460 laney walker blvd, CB-3712
|
| City |
Augusta |
| State/province |
Georgia |
| ZIP/Postal code |
30912 |
| Country |
USA |
| |
|
| Platform ID |
GPL24158 |
| Series (1) |
| GSE126347 |
miRNA profile in human cardiac progenitor cells and iPS cells |
|
