|
| Status |
Public on Aug 31, 2007 |
| Title |
upf1.3 |
| Sample type |
RNA |
| |
|
| Source name |
Neuroblastoma N2A cells
|
| Organism |
Mus musculus |
| Characteristics |
Mouse N2A cells were transfected with Upf1 siRNA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Invitrogen Trizol reagent. RNA samples were quantified using NanoDrop ND-1000 Spectrophotometer.
|
| Label |
biotin
|
| Label protocol |
Total RNA was primed with random hexamers and reverse transcribed. After the reaction was completed, RNA was removed from the reaction by alkaline hydrolysis and the cDNA was purified using Qiagen PCR Quick Purification Kit. A typical reaction started with 5-6ug of total RNA usually yielded ~3ug of cDNA. The cDNA was then fragmented using DNAseI in an empirically controlled reaction that yields DNA fragments of 50-200 bases. This fragmented cDNA was then end labeled using terminal deoxynucleotidyl transferase and "DNA-Labeling-Reagent-1a (DLR-1a)", which is a biotinylated dideoxynucleoside triphosphate.
|
| |
|
| Hybridization protocol |
Targets were hybridized to chips in 7% DMSO solution for 16 hrs overnight at 50. Microarrays were washed, processed with anti-biotin antibodies and streptavidin-phycoerythrin according to the standard Affymetrix protocol.
|
| Scan protocol |
Standard Affymetrix procedures
|
| Description |
Mouse N2A cells were transfected with Upf1 siRNA in order to inhibit nonsense-mediated mRNA decay(NMD) pathway.
|
| Data processing |
Intensity values from the DNA arrays were normalized using a quantile normalization (Bolstad et al., 2003), and probe set summaries were derived using the Robust Multi-chip Analysis (RMA) procedure (Irizarry et al., 2003a; Irizarry et al., 2003b) with two modifications. The first modification was to remove all probes with 17 or more continuous bases that match to any other mouse transcript in order to minimize cross-hybridization issues. The second modification was to use the mode of the probe intensity values of similar GC content probes for the background estimate of a particular probe.
|
| |
|
| Submission date |
Dec 22, 2006 |
| Last update date |
Aug 31, 2007 |
| Contact name |
Manny Ares |
| Organization name |
UCSC
|
| Department |
Molecular and Cellular Biology
|
| Lab |
Ares
|
| Street address |
1125 High St
|
| City |
Santa Cruz |
| State/province |
CA |
| ZIP/Postal code |
95062 |
| Country |
USA |
| |
|
| Platform ID |
GPL2720 |
| Series (1) |
| GSE6611 |
Ultraconserved elements are associated with control of splicing regulators by alt splicing and nonsense mediated decay |
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