|
| Status |
Public on Nov 01, 2013 |
| Title |
H441 transfected with ETS2-specific siRNA 2 |
| Sample type |
RNA |
| |
|
| Source name |
H441 lung cancer cells
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: siRNA against ETS2
cell line: H441
|
| Treatment protocol |
Cells were transfected with control or ETS2-specific siRNA (siRNA from Dharmacon) using Lipofectamine RNAiMAX reagent from Life Technologies according to the manufacturer's instructions.
|
| Growth protocol |
H441 cells were cultured with DMEM/F12 in a humidified incubator with 5CO2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated 48 hours following transfection using the RNeasy Mini Kit from Qiagen according to the manufacturer's instructions.
|
| Label |
Biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 2.5 ug total RNA (Affymetrix).
|
| |
|
| Hybridization protocol |
2.5 ug of fragmented and labeled cDNA, was generated using the Affymetrix GeneChip® WT Terminal Labeling and Controls Kit, were hybridized onto Human Gene 1.0 ST arrays according to the manufacturer’s instructions (Affymetrix). Hybridization cocktails containing samples, control oligonucleotide and eukaryotic hybridization controls in addition to hybridization mixes, DMSO and nuclease-free water were heat denatured at 99oC for 5 minutes, cooled to 45oC for 5 minutes, and finally centrifuged at maximum speed for 1 minute. After injecting 80 uL of the hybridization cocktails, arrays were incubated for 17 hours in a hybridization oven set to a temperature of 45oC with 60 rpm rotation. Arrays were washed, stained and processed using Affymetrix GeneChip Fluidics Station 450 systems
|
| Scan protocol |
Gene chips were scanned using the Affymetrix GeneChip Scanner 3000 7G for subsequent generation of raw data (*CEL files)
|
| Description |
Time of RNA isolation after siRNA transfection is 48 hours
Gene expression of H441 cells transfected with siRNA against the ETS2 gene- biological replicate 2
|
| Data processing |
Raw expression data were normalized and summarized using the Robust Multichip Analysis (RMA) method along with log2 transformation using the integrated BRB-Array Tools software developed by Dr. Richard Simon and the BRB-Array Tools development team.
|
| |
|
| Submission date |
Jan 11, 2013 |
| Last update date |
Nov 01, 2013 |
| Contact name |
Humam Kadara |
| Organization name |
University of Texas MD Anderson Cancer Center
|
| Street address |
2130 W. Holcombe Blvd
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE43459 |
Gene expression profiling of lung cancer cells transfected with scrambled siRNA and siRNA targeting the ETS2 gene |
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