|
| Status |
Public on Jul 29, 2011 |
| Title |
mRNA miR-128-2 (plier) |
| Sample type |
RNA |
| |
|
| Source name |
HEY ovarian cancer cell, miR-128 mimic treated
|
| Organism |
Homo sapiens |
| Characteristics |
originial tumor type of initial cell: Papillary Cystadenocarcinoma of the ovary
cell line: HEY
|
| Biomaterial provider |
Gordon B. Mills
|
| Treatment protocol |
Approximately 12h before transfection, these cells (1.5 × 105) were seeded on six-well plates in growth medium and allowed to adhere overnight at 37°C in a 5% CO2 atmosphere. The following day after washing the wells with PBS and replacing the growth medium with Opti-MEM (Invitrogen) cells were transfected with the miRNA [hsa-miR-7 miRIDIAN mimic, miRIDIAN miRNA mimic negative control #1 (miRNC), or has-miR-128 miRIDIAN mimic [(Thermo Fisher Scientific, Lafayette, CO)] using Lipofectamine 2000 transfection agent (Invitrogen) according to the manufacturer's instructions at a final concentration of 25nM. Cells were incubated for four hours, washed with PBS, and then incubated at 37°C and 5% CO2 for 44h (total 48h) after adding fresh growth medium to the wells.
|
| Growth protocol |
Hey cells were cultured in 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).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated with Trizol (Invitrogen) and purified with PicoPure RNA Isolation Kit (Arcturus). RNA quality was verified on the Bioanalyzer RNA Pico Chip (Agilent Technologies).
|
| Label |
biotin
|
| Label protocol |
mRNAs were converted to double stranded (ds)-cDNA and amplified using Applause 3’-Amp System (NuGen, San Carlos, CA). This cDNA was then biotin labeled and fragmented by using Encode Biotin Module (NuGen)
|
| |
|
| Hybridization protocol |
Labeled mRNA was hybridized to GeneChip Human Genome U133 Plus 2.0 Arrays in the GeneChip Hybridization oven 640, further processed with the GeneChip Fluidics Station 450 and scanned with the GeneChip Scanner 3000.
|
| Scan protocol |
as per manufacturer's protocol using GeneChip Scanner 3000.
|
| Description |
HEY human ovarian carcinoma cell line was derived from a human ovarian cancer xenograft (HX-62) in immunologically deprived CBA/CJ mice originally grown from a peritoneal deposit of a patient.
|
| Data processing |
PLIER via Affymetrix Expression Console (TM) Software Version 1.1. Fold change was calculated based on the log2 difference of average log2 signal values of samples calculated after this preprocessing step.
|
| |
|
| Submission date |
Feb 21, 2011 |
| Last update date |
Jul 29, 2011 |
| 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 |
GPL570 |
| Series (1) |
| GSE27431 |
miRNAs in ovarian cancer: A systems approach (MAS5, plier, GCRMA) |
|
