|
| Status |
Public on Jan 02, 2011 |
| Title |
GB 9 65A NO LPA |
| Sample type |
RNA |
| |
|
| Source name |
NET1 knockdown AGS cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: gastric adenocarcinoma AGS cells
shRNA: 65 shRNA
replicate: A
genotype/variation: NET1 knockdown
agent: no LPA (control)
|
| Treatment protocol |
MISSION™ shRNA Lentiviral Transduction Particles (Sigma Aldrich) were used to achieve stable NET1 knockdown. 5 NET1 specific shRNA constructs, and one non target construct were transduced separately into AGS cells. Briefly, AGS cells were incubated with the Net1-specific shRNA lentiviral particles at a ratio of 2 particles : 1 cell, in the presence of hexadimethrine bromide to improve transduction efficiency. Non-target shRNA lentiviral particles were used to control for the effects of the transduction process itself. Successfully transduced cells included a puromycin resistance tag, and these cells were selected using 1μg/ml puromycin. Net 1 knockdown was confirmed using quantitative RTPCR and western blot analysis. For the remainder of this study, two distinct cell types were used: NET1 knockdown cells and non-target shRNA cells.
|
| Growth protocol |
Human Caucasian gastric adenocarcinoma cells (AGS cells) were purchased from the European Collection of Cell Cultures (ECACC) and cultured in Hams F12 medium (Sigma Aldrich), supplemented with 10% fetal bovine serum, L-glutamine and Penicillin-Streptomycin (Sigma Aldrich), as per ECACC recommendations and incubated at 37ºC and 5%CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extraction by Trizol™ (Sigma Aldrich) following the manufacturers instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 μg total RNA (Expression Analysis Technical Manual, 2001, Affymetrix) using the Affymetrix IVT labeling kit.
|
| |
|
| Hybridization protocol |
Following fragmentation, cRNA was hybridized for 16 hr at 45C on Affymetrix U133 plus 2.0 genechips. GeneChips were washed and stained in the Automated fluidics 450 station.
|
| Scan protocol |
Genechips were scanned using the GeneChip 3000 7G scanner
|
| Description |
Gene expression in NET1 knockdown AGS cells.
GB 9 65A NO LPA.CEL
|
| Data processing |
Scanned images, obtained using Affymetrix Software (MAS5), were normalized using robust multichip average (RMA) express analysis
|
| |
|
| Submission date |
Dec 27, 2010 |
| Last update date |
Jan 02, 2011 |
| Contact name |
David MURRAY |
| Organization name |
RCSI
|
| Street address |
123 St Stephens Green
|
| City |
Dublin |
| ZIP/Postal code |
D2 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL571 |
| Series (1) |
| GSE26309 |
A Transcriptomic Analysis of NET1 (a RhoA GEF Exchange Factor) in AGS Gastric Cancer Cells |
|
