|
| Status |
Public on Jun 30, 2012 |
| Title |
knockout rep-3 |
| Sample type |
RNA |
| |
|
| Source name |
Mouse soleus
|
| Organism |
Mus musculus |
| Characteristics |
tissue: skeletal muscle
strain: mixed background (C57Bl/6 and 129)
genotype: Yin Yang 1 (YY1) knockout
age: 6 months
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
The double-stranded cDNA was used as the template in an in vitro transcription (IVT) reaction catalyzed by T7 polymerase and containing biotinylated CTP and UTP in addition to the four unmodified ribonucleoside triphosphates. The biotinylated complementary RNA (cRNA) waas purified from the IVT reaction mixture using magnetic particles. The cRNA was quantified spectrophotometrically and purity of the cRNA is also assessed by spectrophometric measurements. The purified cRNA was fragmented in order to facilitate the subsequent hybridization step. Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
|
| |
|
| Hybridization protocol |
The fragmented cRNA was added to a hybridization solution containing several biotinylated control oligonucleotides (for quality control), and hybridized to a microarray chip overnight at 45°C. The hybridization solution was then removed. The chips were transferred to a fluidics instrument that performs washes to remove cRNA that has not hybridized to its complementary oligonucleotide probe. The bound cRNA was then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE); additional fluors are then added using biotinylated anti-streptavidin antibody and additional SAPE.
|
| Scan protocol |
Each cRNA bound at its complementary oligonucleotide was excited using a confocal laser scanner, GCS3000, and the positions and intensities of the fluorescent emissions were captured.
|
| Description |
Gene expression data from knockout soleus
|
| Data processing |
The data were analyzed with RMA (Gene Pattern's ExpressionFileCreator version 8). Parameters: method RMA quantile normalization yes background correct yes compute present absent calls no normalization method none value to scale to clm file annotate probes yes The data were analyzed with RMA (Gene Pattern's ExpressionFileCreator version 8). Parameters: method RMA quantile normalization yes background correct yes compute present absent calls no normalization method none value to scale to clm file annotate probes yes.
|
| |
|
| Submission date |
Jun 28, 2012 |
| Last update date |
Jun 30, 2012 |
| Contact name |
Pere Puigserver |
| E-mail(s) |
pere_puigserver@dfci.harvard.edu
|
| Phone |
617-582-7977
|
| Organization name |
Dana-Farber Cancer Institute
|
| Street address |
450 Brookline Av. CLSB-11144
|
| City |
Boston |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL8321 |
| Series (1) |
| GSE39009 |
Expression data from mouse skeletal muscle |
|
