|
| Status |
Public on Aug 15, 2008 |
| Title |
HeLa vs pool |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HeLa
|
| Organism |
Homo sapiens |
| Characteristics |
48 diverse human tissues and cell lines were hybridized to a 17-array set. Samples were purchased as pools from multiple donors, typically over 10 (Clontech, Mountainview, CA).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA isolated using Qiagen RNeasy spin columns with DNAse treatment; polyA+ RNA isolated from total RNA via magnetic bead-based mRNA extraction (Ambion, Poly(A) Purist)
|
| Label |
Cy3
|
| Label protocol |
custom automated version of the aminoallyl MessageAmp II kit from Ambion.
|
| |
|
| Channel 2 |
| Source name |
pool
|
| Organism |
Homo sapiens |
| Characteristics |
Pooled RNA from 20 diverse disease-free adult tissue pools comprised the reference pool.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA isolated using Qiagen RNeasy spin columns with DNAse treatment; polyA+ RNA isolated from total RNA via magnetic bead-based mRNA extraction (Ambion, Poly(A) Purist)
|
| Label |
Cy5
|
| Label protocol |
custom automated version of the aminoallyl MessageAmp II kit from Ambion.
|
| |
|
| |
| Hybridization protocol |
Microarrays are incubated at 40°C for 48 hours in a rotating carousel. Hybridizations to custom Agilent microarrays are completed as previously described (Hughes et al.Nat Biotech (2001), 19(4):342-7). Microarrays are washed to remove non-specific hybridized sample. Afterwards, microarrays are dried in an ozone-free nitrogen chamber.
|
| Scan protocol |
Microarrays are scanned using the Agilent LP2 laser scanner. The scanner output is a Tiff file, which contains the quantitative hybridization data from each individual microarray. The Tiff files are then processed using Rosetta custom feature extraction software.
|
| Description |
HeLa vs pool
|
| Data processing |
Data were processed using the Rosetta Resolver® system. Rosetta's custom feature extraction software performs error modeling before data are loaded into the Resolver system. The Resolver system performs a squeeze operation that combines replicates of the same sequence in an array while applying error weighting. The error weighting consists of adjusting for additive and multiplicative noise. A P-value is generated and propagated throughout the system. The P-value represents the probability that a gene is expressed. The Resolver system allows users to set thresholds, below which genes of a P-value are considered to be significantly expressed. The Resolver system also combines multiple arrays using a squeezing process. If multiple spots reference one sequence, summarization is performed using an error-weighted average as described in Roland Stoughton and Hongyue Dai, Statistical Combining of Cell Expression Profiles. US Patent #6,351,712, February 26, 2002.
|
| |
|
| Submission date |
Jun 02, 2008 |
| Last update date |
Jul 28, 2008 |
| Contact name |
Amit V Kulkarni |
| E-mail(s) |
amit_kulkarni@merck.com
|
| Phone |
206-802-7352
|
| Organization name |
Rosetta Inpharmatics / Merck Pharmaceuticals
|
| Department |
Genomics
|
| Lab |
Array Design
|
| Street address |
401 Terry Ave N
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL6833 |
| Series (1) |
| GSE11863 |
Differential expression of 24,426 human alternative splicing events and predicted cis-regulation in 48 tissues |
|
