|
| Status |
Public on Apr 10, 2013 |
| Title |
MCF7_RHBDD2siRNA_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
MCF7_RHBDD2siRNA_rep2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
tissue: breast adenocarcinoma
|
| Treatment protocol |
MCF7 and T47D cell lines were cultured on a 12 well plate at 40% of confluence in Opti-MEM I Reduced Serum Medium, and were transiently transfected with 40 pmoles/ul of siRNA mixed with Lipofectamine according to the manufacturer's protocol (Invitrogen, USA).
|
| Growth protocol |
Estrogen dependent breast cancer cell lines MCF7 and T47D were cultured in RPMI medium (GIBCO, Gaithersburg, MD) and DMEM (GIBCO, Gaithersburg, MD) respectively, supplemented with 10% FBS (Bioser, Argentina), 10 U/ml penicillin G and 10 μg/ml streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were isolated and purified using the TRIreagent and Nucleo Spin RNA Clean-up Kit (Macherey-Nagel) from two breast cancer cell lines (MCF7 and T47D) derived from the negative control-siRNA and the RHBDD2-siRNA treatments in duplicate experiments. RNA concentration and integrity were measured on Agilent Bioanalyzer RNA 6000 Nanochip (Agilent Technologies).
|
| Label |
Cy5
|
| Label protocol |
Aminoallyl-amplified RNA were synthesized from one microgram of total RNA with the Amino Allyl MessageAmpTM II aRNA Amplification kit (Ambion) and subsequently labeled with Cy5 Mono-ReactiveDyePack (GE Health Care Bioscience).
|
| |
|
| Hybridization protocol |
Ten micrograms of labeled RNA were probed (during 16hrs, 37°C, 250 rpm) using the whole genome Toray 3D-GeneTM Human Oligo Chip 25k V2.1.
|
| Scan protocol |
The arrays were scanned with a Toray 3D-GeneTM Scanner.
|
| Description |
human oligo chip 25k ver.2.1
|
| Data processing |
TIFF images of all arrays were quantified using ExtractionTM Software (Toray). For genes of which the signal was more than Background mean + 2SD, the raw signal was subtracted with the Background mean. Data were normalized to the level in which the median value was 25.
|
| |
|
| Submission date |
Dec 19, 2012 |
| Last update date |
Apr 10, 2013 |
| Contact name |
Martin Carlos Abba |
| E-mail(s) |
mcabba@gmail.com
|
| Phone |
054-221-4236711
|
| Organization name |
School of Medical Sciences - UNLP
|
| Lab |
CINIBA
|
| Street address |
60 y 120
|
| City |
La Plata |
| State/province |
Buenos Aires |
| ZIP/Postal code |
1900 |
| Country |
Argentina |
| |
|
| Platform ID |
GPL13915 |
| Series (1) |
| GSE43015 |
RHBDD2 silencing and gene expression profiling of breast cancer cells |
|
