|
| Status |
Public on Apr 09, 2012 |
| Title |
miRNA HEY 2 |
| Sample type |
RNA |
| |
|
| Source name |
Cultured HEY epithelial ovarian carcinoma cell line, pre-transfected
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEY
cell type: epithelial ovarian carcinoma
treatment: pre-transfected
|
| Treatment protocol |
Approximately 16h after seeding, the wells were washed with PBS and the growth medium was replaced with Opti-MEM (Invitrogen) to simulate identical conditions of transfection. Following 4 hours of incubation in the reduced serum Opti-MEM medium, the cells were switched back to complete growth medium (RPMI 1640) and allowed to grow for another 48 hours before RNA extraction.
|
| Growth protocol |
The cells (1.5 × 10^5 per well) were seeded on six-well plates in growth medium RPMI 1640 (Mediatech, Manassas, VA) supplemented with 10 % v/v heat-inactivated fetal calf serum (Invitrogen, Carlsbad, CA), 2 mM L-glutamine (Mediatech), 10 mM HEPES buffer (Mediatech), penicillin (100 U/ml), and streptomycin (100 μg/mL) and were allowed to adhere overnight at 37°C in a 5 % CO2 atmosphere.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from the pre-transfected HEY cells using the mirVana miRNA isolation kit according to the manufacturer's instructions (Applied Biosystems, Foster City, CA).
|
| Label |
biotin
|
| Label protocol |
MiRNAs were labeled with the Genisphere FlashTag HSR Biotin RNA labeling kit (Genisphere, Hatfield, PA).
|
| |
|
| Hybridization protocol |
Hybridization with GeneChip miRNA Array chips (Affymetrix, Santa Clara, CA) was carried out according to the manufacturer's instructions.
|
| Scan protocol |
Scanning was performed according to Affymetrix-recommended procedures using the GeneChip scanner 3000.
|
| Description |
Biological replicate 2 of 2.
|
| Data processing |
Raw data in the form of CEL files were produced by the Affymetrix GeneChip Operating System (GCOS) software and analyzed using miRNA QC Tool software (Affymetrix). Normalization was performed using the default workflow of the miRNA QC Tool software. Briefly, probesets were tested for detection above background using a Wilcoxon Rank-Sum test (p <0.06) and then normalized using quantile normalization. Probesets with ‘FALSE’ call in all groups were removed from statistical analysis. A constant (16) is added for variance stabilization and then values are summarized using the median polish method. The log base 2 transformed expression values were then used for calculations.
|
| |
|
| Submission date |
May 06, 2011 |
| Last update date |
Apr 09, 2012 |
| Contact name |
Shubin Shahab |
| E-mail(s) |
shubin.shahab@gmail.com
|
| Organization name |
Georgia Institute of Technology
|
| Department |
Biology
|
| Lab |
McDonald
|
| Street address |
310 Ferst Drive
|
| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30332 |
| Country |
USA |
| |
|
| Platform ID |
GPL8786 |
| Series (1) |
| GSE29126 |
miRNA expression profile of pre-transfected HEY cells |
|
