|
| Status |
Public on Oct 01, 2011 |
| Title |
macrophage_m29_C57BL/6-Ldlr-/- |
| Sample type |
RNA |
| |
|
| Source name |
macrophages
|
| Organism |
Mus musculus |
| Characteristics |
tissue: n/a
cell type: macrophage
genetic background: C57BL/6
genotype: C57BL/6-Ldlr-/-
|
| 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 reverse 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-cDNA.
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| |
|
| 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 |
Common genetic variants in a 58-kilobase (kb) region of chromosome 9p21, near the CDKN2A/B cell proliferation inhibitor genes, are strongly associated with coronary artery disease (CAD). We previously reported a congenic mouse model harboring an atherosclerosis susceptibility locus in a region of homology with the human 9p21 locus. We now report markedly decreased Cdkn2a (cyclin-dependent kinase inhibitor 2a) mRNA expression in macrophages and increased circulating levels of Ly6Chigh inflammatory monocytes in congenic mice compared to controls. A bone marrow (BM) transplantation study revealed that BM-derived cells from Cdkn2a+/- mice are capable of conferring accelerated atherosclerosis, increased inflammatory monocyte subsets and monocyte proliferation in the Ldlr-/- mouse model. These findings provide a mechanistic link between decreased expression of Cdkn2a transcripts, increased monocyte proliferation, and accelerated atherosclerosis.
|
| 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).
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| |
|
| Submission date |
Sep 09, 2010 |
| Last update date |
Oct 01, 2011 |
| Contact name |
Oscar Puig |
| E-mail(s) |
[email protected]
|
| Organization name |
Merck Research Laboratories
|
| Department |
Molecular Profiling Research Informatics
|
| Street address |
126 East Lincoln Ave
|
| City |
Rahway |
| State/province |
NJ |
| ZIP/Postal code |
07065 |
| Country |
USA |
| |
|
| Platform ID |
GPL9734 |
| Series (1) |
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