|
| Status |
Public on Nov 02, 2012 |
| Title |
P5.221EA5_SmoA2 mutant |
| Sample type |
RNA |
| |
|
| Source name |
Brain
|
| Organism |
Mus musculus |
| Characteristics |
tissue: brain
strain: C57BL/6
genotype/variation: P5.221EA5_SmoA2 mutant
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples are processed in parallel in 96-well plates to minimize potential variation. Reaction purification is achieved using magnetic binding beads for cDNA and Qiagen RNeasy kits for cRNA purification.
|
| Label |
biotin
|
| Label protocol |
Biotin is incorporated during the amplification. This binds a streptavidin-conjugated phycoerythrin during the post-hybridization staining process that provides the measurable signal.
The final in vitro transcription incorporates biotin moieties that are later labeled with phycoerythrin. After amplification, samples are put through a controlled fragmentation to improve hybridization sensitivity and consistency. The labeled molecules are biotinylated-cRNA.
|
| |
|
| Hybridization protocol |
GeneChip microarrays are loaded with the fragmented target sample/hybridization buffer mix using standard manual techniques. Arrays are hybridized for 18 hours at 45'C with vigorous mixing. Unbound sample is removed and staining is accomplished through the binding of streptavidin conjugated phycoerythrin to the hybridized target. Excess label is removed. Washing and staining steps are performed by the Affymetrix FS450 fluidics station using standard protocols.
|
| Scan protocol |
Arrays are scanned using a GeneChip Scanner 3000 7G with a 48 array autoloader. The scanner maintains the optimal temperature for the arrays prior to and during scanning.
|
| Description |
Affymetrix gene expression profiling of mouse Smoothened mutant and wild type samples
|
| Data processing |
Data were loaded into the Rosetta Resolver' system (Rosetta Biosoftware, Seattle, WA) and processed using the RMA algorithm (http://www.ncbi.nlm.nih.gov/sites/entrez'cmd=Retrieve&db=PubMed&list_uids=12582260). Intensities were calculated based on RMA, a Rosetta-developed error, and a MAS-5 p-value. The Rosetta-developed error is used in the calculation of xdev for the ratio p-values as described in section 2.2 (http://bioinformatics.oxfordjournals.org/cgi/content/full/22/9/1111).
|
| |
|
| Submission date |
Nov 28, 2011 |
| Last update date |
Nov 02, 2012 |
| Contact name |
Michele Cleary |
| E-mail(s) |
[email protected]
|
| Phone |
215 652-6104
|
| Organization name |
Merck and Co., Inc.
|
| Department |
Genetics and Pharmacogenomics
|
| Lab |
Target & Pathway Biology
|
| Street address |
770 Sumneytown Pike
|
| City |
West Point |
| State/province |
PA |
| ZIP/Postal code |
19486 |
| Country |
USA |
| |
|
| Platform ID |
GPL9734 |
| Series (1) |
| GSE34593 |
Oncogenic mutation in Smoothened causes severe cerebellar developmental defects and medulloblastoma in mice |
|
