|
| Status |
Public on Feb 15, 2010 |
| Title |
MDA IgG pulldown rep2 |
| Sample type |
other |
| |
|
| Source name |
MDA
|
| Organism |
Homo sapiens |
| Characteristics |
ip: IgG
er status: -
cell line: MDA-MB-231
|
| Extracted molecule |
total RNA |
| Extraction protocol |
HuR Immunoprecipitations (RIP-Chip): Cell lysates were prepared from exponentially growing MB-231 and MCF-7 cells. Equal amounts of protein lysates were used (100-300μg). HuR monoclonal antibody 3A2 or isotype control IgG1 were pre-coated onto protein A Sepharose beads (PAS) and extensively washed. Lysates from each cell line initially were pre-absorbed with 30 μg of IgG1 and then removed by addition of PAS beads. Individual pull downs were performed at 4°C for only 1-2 hr to minimize potential re-assortment of mRNAs.
|
| Label |
biotin
|
| Label protocol |
The entire amount of recovered RNA per immunoprecipitation was amplified using the WT-Ovation™ Pico RNA Amplification System. Forty ng of total RNA was used as starting material to generate at least 6μg of cDNA. Amplified cDNA was purified and then incubated at 50°C for 30 minutes with 5μl of UNG buffer and 5μl UNG enzyme and 60 minutes with 5μl labeling buffer and 5μl ARP (biotin) solution as described in NuGen’s labeling protocol for the Illumina Beadarray platform. All samples were quantitated, and RNA quality and integrity were assessed on selected samples with the Experion™ automated electrophoresis system.
|
| |
|
| Hybridization protocol |
Biotin-labeled, amplified cDNA (1.5µg) was hybridized to a Sentrix® Human-6 v.2 Whole Genome Expession BeadChips. The chips were hybridized at 48°C for 20 hr in the hybridization buffer provided by the manufacturer. After hybridization, the chips were washed and stained with streptavidin-C3.
|
| Scan protocol |
The chips were scanned on the BeadArray Reader, as described by Illumina at http://www.illumina.com.
|
| Description |
replicate 2
|
| Data processing |
The Illumina Beadstudio software was used to assess fluorescent hybridization signals. The data were background adjusted with lumi in R using the 'forcePositive' method, and then log base 2 transformed. The data were then normalized using quantile normalization with lumi in R.
|
| |
|
| Submission date |
Aug 26, 2009 |
| Last update date |
Feb 11, 2010 |
| Contact name |
J Wade Davis |
| Organization name |
AbbVie
|
| Street address |
1 N Waukegan Rd
|
| City |
North Chicago |
| State/province |
IL |
| ZIP/Postal code |
60048 |
| Country |
USA |
| |
|
| Platform ID |
GPL7363 |
| Series (1) |
| GSE17820 |
HuR differentially regulates unique subsets of mRNAs in estrogen receptor negative and positive breast cancer |
|
