|
Status |
Public on Jul 19, 2010 |
Title |
EPT294 ependynoma [human miRNA] |
Sample type |
RNA |
|
|
Source name |
patient sample
|
Organism |
Homo sapiens |
Characteristics |
disease state: ependynoma primary tumor age: 7 Sex: F
|
Treatment protocol |
tumors excised and flash frozen at distinct sites, storage and freezing method varies
|
Extracted molecule |
total RNA |
Extraction protocol |
standard Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
Label |
Cy3
|
Label protocol |
Cyanine-3 labeled miRNA was prepared from 100 ng total RNA using the Agilent miRNA Microarray System with miRNA Complete Labeling and Hyb Kit, v.2.0 (June 2008) according to the manufacturer's instructions. No post labeling purification was performed, followed by vacuum drying of the labeled reactions, per the protocol.
|
|
|
Hybridization protocol |
Per the protocol, the dried labeled product was resuspended in nuclease-free water, followed by addition of Agilent GE Blocking Agent and Agilent Hi-RPM Hybridization Buffer. The labeled miRNA was then heated to 100°C for 5 minutes, followed by 5 minutes in an ice-water bath. The entire volume (45 µl) of eight, individual reactions was hybridized to Human 8x15K miRNA Microarrays (V2), Agilent #G4470B, for 20 hours at 55°C, 20 rpm, 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 by brief centrifugation.
|
Scan protocol |
Slides were immediately scanned after washing on the Agilent Microarray Scanner (G2565CA) using one color scan setting for 8x15k array slides, using the following scan settings: 20-bit, scan area 61x21.6 mm, single pass, scan resolution 5 µm, 100% PMT, green dye channel only, and eXtended Dynamic range (XDR) not enabled
|
Description |
analyzed at St. Jude Hartwell Center
|
Data processing |
The scanned images were analyzed with Feature Extraction Software 10.1 (Agilent) using default parameters (protocol miRNA_105_Dec08 and Grid: 019118_D_20080215) to obtain background detrended processed signal intensities, which was then normalized, scaled and averaged. The minimum detecting miRNA expression signal was set at a threshold with less than 1% of the negative control probes.
|
|
|
Submission date |
May 05, 2010 |
Last update date |
Oct 08, 2010 |
Contact name |
Richard James Gilbertson |
E-mail(s) |
richard.gilbertson@stjude.org
|
Organization name |
St Jude Children's Research Hospital
|
Department |
Developmental Neurobiology
|
Street address |
262 Danny Thomas Place
|
City |
Memphis |
State/province |
TN |
ZIP/Postal code |
38105 |
Country |
USA |
|
|
Platform ID |
GPL8227 |
Series (1) |
GSE21687 |
Comparative genomics matches mutations and cells to generate faithful ependymoma models |
|