|
| Status |
Public on Aug 01, 2015 |
| Title |
ES-7 [miRNA] |
| Sample type |
RNA |
| |
|
| Source name |
Ewing Sarcoma
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: ES-7
cell type: human Ewing Sarcoma cell line
|
| Growth protocol |
Immortalized human cell lines derived from sarcoma's were purchased from ATCC or provided by Memorial Sloan Kettering Cancer Center , Children's Oncology Group, and Pediatric Preclinical Testing Program, and grown in their recommended media supplemented with 10-20% fetal bovine serum. Flasks of cells in media were maintained in incubators with 95% air and 5% CO2 at 37 degrees C, and passaged as the cells reached 70-90% confluency.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were grown in flasks to approximately 70% confluency, detatched with trypsin, and then total RNA (including miRNA) extracted using Qiagen miRNeasy mini-kit according to manufacturers instructions. Quality of RNA was assessed using an Agilent 2100 Bioanalyzer and all samples were of good quality with RIN# > 8.
|
| Label |
fluorescence
|
| Label protocol |
Total RNA (100 ng) was ligated to unique oligonuceotide tag to increase the length of the short miRNA for subsequent detection in a single reaction without amplification using NanoString kit following manufacturer’s suggested protocol.
|
| |
|
| Hybridization protocol |
Samples were hybridized for 16 hrs to NanoString human miRNA probeset, which contains probe pairs specific for each miRNA with different fluorescent barcode labels. The probe pair consists of a Reporter Probe, which carries the fluorescent signal on its 5' end, and a Capture Probe which carries a biotin on its 3' end. After hybridization, excess probes are washed away using a two step magnetic bead-based purification on the nCounter™ Prep Station and are immobilized in the cartridge for data collection.
|
| Scan protocol |
Data Collection is carried out in the nCounter Digital Analyzer. Digital images are processed and the barcode counts are tabulated in a RCC format.
|
| Data processing |
Data were analyzed using custom R scripts
|
| |
|
| Submission date |
Jun 02, 2015 |
| Last update date |
Aug 01, 2015 |
| Contact name |
Eric C Polley |
| E-mail(s) |
eric.polley@nih.gov
|
| Organization name |
National Cancer Institute
|
| Department |
Biometric Research Branch
|
| Street address |
9609 Medical Center Drive, Rm 5W118
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL20275 |
| Series (2) |
| GSE69470 |
microRNA expression profiling of human sarcoma cell lines (~68) in the Molecular Pharmacology Branch, DTP, DCTD NCI collection of human cell lines |
| GSE69524 |
NCI Sarcoma Cell Line Panel |
|
