|
| Status |
Public on Jan 30, 2010 |
| Title |
ATF4 transfection, experiment 4 |
| Sample type |
RNA |
| |
|
| Source name |
RNA from skeletal muscle transfected with plasmid encoding mouse ATF4
|
| Organism |
Mus musculus |
| Characteristics |
age: 8 weeks
sex: male
tissue: tibialis anterior
|
| Treatment protocol |
Skeletal muscles were transfected in vivo with plasmid DNA, and RNA was harvested from transfected muscles (under nonfasting conditions) 11 days later.
|
| Growth protocol |
Not applicable
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol solution (Invitrogen) according to the manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
50 ng total RNA was converted to SPIA amplified cDNA using the WT-Ovation Pico RNA Amplification System, v1 (NuGEN Technologies, San Carlos, CA, Cat. #3300) according to the manufacturer’s recommended protocol. The amplified SPIA cDNA product was purified through a QIAGEN MinElute Reaction Cleanup column (QIAGEN Cat #28204) according to modifications from NuGEN. Four μg of SPIA amplified DNA were used to generate ST-cDNA using the WT-Ovation Exon Module v1 (NuGEN Technologies, Cat #2000) and again cleaned up with the Qiagen column as above. Five μg of this product were fragmented (average fragment size = 85 bases) and biotin labeled using the NuGEN FL-Ovation cDNA Biotin Module, v2 (NuGEN Technologies, Cat. #4200) per the manufacturer’s recommended protocol.
|
| |
|
| Hybridization protocol |
Biotin-labeled cDNA was mixed with Affymetrix eukaryotic hybridization buffer (Affymetrix, Inc., Santa Clara, CA), placed onto Mouse Exon 1.0 ST arrays, and incubated at 45º C for 18 h with 60 rpm rotation in an Affymetrix Model 640 Genechip Hybridization Oven.
|
| Scan protocol |
Following hybridization, the arrays were washed, stained with streptavidin-phycoerythrin (Molecular Probes, Inc., Eugene, OR), signal amplified with antistreptavidin antibody (Vector Laboratories, Inc., Burlingame, CA) using the Affymetrix Model 450 Fluidics Station. Arrays were scanned with the Affymetrix Model 3000 scanner with 7G upgrade and data were collected using the using the GeneChip operating software (GCOS) v1.4.
|
| Description |
RNA from skeletal muscle transfected with plasmid encoding mouse ATF4
|
| Data processing |
RMA normalization
HuEx-1_0-st-v2.r2.pgf
HuEx-1_0-st-v2.r2.d1.1.hg18.core.mps
|
| |
|
| Submission date |
Jan 29, 2010 |
| Last update date |
Jan 29, 2010 |
| Contact name |
Christopher Adams |
| Organization name |
University of Iowa
|
| Street address |
540G EMRB, 200 Hawkins Drive
|
| City |
Iowa City |
| State/province |
IA |
| ZIP/Postal code |
52242 |
| Country |
USA |
| |
|
| Platform ID |
GPL6096 |
| Series (2) |
| GSE20101 |
Effect of ATF4 overexpression on mouse skeletal muscle mRNA levels |
| GSE20104 |
Determining the mechanism of ATF4-mediated myofiber atrophy |
|
