|
| Status |
Public on Jun 15, 2008 |
| Title |
primary OB #2 |
| Sample type |
RNA |
| |
|
| Source name |
osteoblasts (in vitro differentiated)
|
| Organism |
Mus musculus |
| Characteristics |
WT
|
| Growth protocol |
mouse model
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol/QIAGEN columns
|
| Label |
biotin
|
| Label protocol |
RNA is converted into cDNA using a T7 promoter-tailed oligo-dT primer in the synthesis of the first cDNA strand; second strand cDNA synthesis is then carried out. The double-stranded cDNA is 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) is purified from the IVT reaction mixture using the RNeasy system (Qiagen). The cRNA is quantified spectrophotometrically and purity of the cRNA is also assessed by spectrophometric measurements. Those cRNA's that fall outside of an acceptable range will not be carried forward in the analysis. Should this occur, investigators would be notified and asked to provide a new RNA sample. The purified cRNA is fragmented in order to facilitate the subsequent hybridization step. (http://chip.dfci.harvard.edu/lab/services.php)
|
| |
|
| Hybridization protocol |
The fragmented cRNA is added to a hybridization solution containing several biotinylated control oligonucleotides (for quality control), and hybridized to a microarray chip overnight at 45°C. The chips are then transferred to a fluidics instrument that performs washes to remove cRNA that has not hybridized to its complementary oligonucleotide probe. The bound cRNA is then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE); additional fluors are then added using biotinylated anti-streptavidin antibody and additional SAPE. (http://chip.dfci.harvard.edu/lab/services.php)
|
| Scan protocol |
Each cRNA bound at its complementary oligonucleotide is excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions are captured. These measures provide the basis of subsequent biostatistical analysis. (http://chip.dfci.harvard.edu/lab/services.php)
|
| Description |
determine the expression profile of mouse osteosarcoma
|
| Data processing |
dChip normalization
|
| |
|
| Submission date |
Oct 30, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Carl Walkley |
| E-mail(s) |
cwalkley@svi.edu.au
|
| Organization name |
Dana-Farber Cancer Institute
|
| Department |
Pediatric Oncology
|
| Street address |
44 Binney St
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE9460 |
Mouse model of Osteosarcoma |
|
| Relations |
| Reanalyzed by |
GSE119085 |
