|
| Status |
Public on Jun 09, 2010 |
| Title |
HeLa, siRNA, EZH2-6, 0.5nM, 24hr |
| Sample type |
RNA |
| |
|
| Source name |
Hela cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Hela cells
conditions: siRNA, EZH2-6, 0.5nM, 24hr
|
| 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 |
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 Hela profiling of siRNA/shRNA treatments
|
| 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 |
Jun 07, 2010 |
| Last update date |
Jun 08, 2010 |
| Contact name |
Michele Cleary |
| E-mail(s) |
michele_cleary@merck.com
|
| 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 |
GPL10379 |
| Series (1) |
| GSE22209 |
Reduced seed region-based off-target activity with lentivirus-mediated RNAi |
|
