|
| Status |
Public on Jun 30, 2008 |
| Title |
OVCAR5 |
| Sample type |
RNA |
| |
|
| Source name |
Ovary
|
| Organism |
Homo sapiens |
| Characteristics |
Ovarian serous carcinoma (ascites)
Gender: female
|
| Treatment protocol |
None
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TRIZOL RNA preparation (Invitrogen)
|
| Label |
Single Channel Hy3 fluorescent label (Exiqon miRCURY LNA Labeling Kit)
|
| Label protocol |
1 ug of total RNA was 3' end labeled with Hy3 following the Exiqon miRCURY LNA Labeling Kit protocol. In short, 1 ug of total RNA and spike-in miRNA from the Exiqon kit was mixed with labeling buffer and labeling enzyme and incubated at 0 degrees C for 1 hour in a PCR machine. The reaction was stopped by incubating 15 minutes at 65 degrees C. The samples was then ready for hybridization.
|
| |
|
| Hybridization protocol |
The labeled sample was diluted to 40ul, added to 40ul of 2X hybridization buffer and incubated at 95 degrees C for 4 minutes. Samples were then cooled by a 2 minute spin at the max speed in a microcentrifuge. Die-tech hyb chambers were prepared with ~30ul of 4X salt buffer in the humidity wells and the Exiqon microRNA arrays were loaded and covered with LifterSlips(25X50I-M-5597-001-LS). The entire sample was loaded under the lifterslip, the chambers were closed tightly and placed in a 60 degree water bath for 16-18 hours. The arrays are removed from the chambers, washed once at 60 degrees in wash buffer A for 2 minutes which removed the lifterslip, and then twice at room temperature in wash buffer B, first for 10 seconds to remove all detergent present from the first wash, then for 2 minutes in fresh wash B. Finally the slide was placed in wash C for 2 minutes at room temperature, then dried by spinning at 500 RPM for 4 minutes.
|
| Scan protocol |
Slides were scanned in an Agilent scanner at 10um resolution and the spot intensity data was extracted with Agilent Feature Extractor software v7.5.
|
| Description |
microRNA expression data generated from Exiqon miRCURY LNA microRNA Arrays hybridized and scanned using a single channel of Hy3 fluorescent label.
|
| Data processing |
The 4 replicate spot intensities per array were averaged in an excel spreadsheet to give the 1600 results in this file. Then the results were normalized using a global normalization of the total average intensity of all non-control probe intensities from each array as compared to the average intensities from all of the arrays. i.e. [ (intensity of spot #1 from array A) X (average intensity from all arrays / average intensity of array A) ]. Differently expressed microRNA species were identified between the different sample groups by the ratio of these normalized intensities.
|
| |
|
| Submission date |
Jan 11, 2008 |
| Last update date |
Jun 22, 2020 |
| Contact name |
Supriyo De |
| Organization name |
NIA-IRP, NIH
|
| Department |
Laboratory of Genetics and Genomics
|
| Lab |
Computational Biology & Genomics Core
|
| Street address |
251 Bayview Blvd
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21224 |
| Country |
USA |
| |
|
| Platform ID |
GPL6350 |
| Series (1) |
|
