|
| Status |
Public on Jun 21, 2010 |
| Title |
DIO_CB1R-/-_KO_10mpk_AM251_Liver_47 |
| Sample type |
RNA |
| |
|
| Source name |
liver
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
diet: high fat
genotype/variation: CB1R-/- (KO)
treatment: 10mpk AM251
tissue: liver
|
| 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 is incorporated during the amplification. This binds a streptavidin-conjugated phycoerythrin during the post-hybridization staining process that provides the measurable signal.
|
| Label protocol |
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 |
DIO CB1R-/- KO vehicle (n=4) Virtual Pool vs. DIO_CB1R-/-_KO_10mpk_AM251_Liver_47
|
| Data processing |
Data were processed using the Rosetta Resolver® system. The software performs error modeling using the data loaded and processed into the Resolver system. The Resolver system applies the RMA algorithm to combine replicates of the same sequence in an array. Affymetrix's MAS-5 method is applied to calculate a P-value for such sequences. The P-value represents the probability that a gene is expressed. The Resolver system allows users to set thresholds of a P-value below which, genes are considered to be significantly expressed. Errors for the RMA-computed sequences are fitted using methodology developed by Rosetta using the guidelines set in Weng et al. (2006) Bioinformatics 22:1111-1121. The Resolver system also combines multiple arrays using an averaging process. The P-values for the combined results are also the combination of individual probe-level P-values (geometric means). Ratios and their P-values are computed as described in section 2.2 of Weng et al. (2006).
|
| |
|
| Submission date |
Mar 25, 2010 |
| Last update date |
Jun 21, 2010 |
| Contact name |
Olivia Fong |
| E-mail(s) |
olivia.fong@merck.com
|
| Organization name |
Merck & Co.
|
| Department |
Molecular Profiling
|
| Lab |
Merck Research Laboratories
|
| Street address |
P.O. Box 2000
|
| City |
Rahway |
| State/province |
NJ |
| ZIP/Postal code |
07065 |
| Country |
USA |
| |
|
| Platform ID |
GPL9734 |
| Series (1) |
| GSE21069 |
Genome-wide expression profiling revealed peripheral effects of CB1 inverse agonists in improving insulin sensitivity and metabolic parameters |
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