|
| Status |
Public on Jan 22, 2010 |
| Title |
MRK025_Stroma |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Stratagene Universal Human Reference RNA
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Reference RNA Pool
|
| Treatment protocol |
Human tissue specimens were surgically excised and snap frozen in liquid nitrogen
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Messenger RNA was extracted from stromal cells microdissected from 5x 8 μm sections using PicoPureRNA reagents according to Molecular Devices' instructions
|
| Label |
Cy3
|
| Label protocol |
Custom automated version of the aminoallyl MessageAmp II kit from Ambion. 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.
|
| |
|
| Channel 2 |
| Source name |
MRK025_Stroma
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: stromal cells (laser-capture microdissected)
disease stage: Adenocarcinoma
|
| Treatment protocol |
Human tissue specimens were surgically excised and snap frozen in liquid nitrogen
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Messenger RNA was extracted from stromal cells microdissected from 5x 8 μm sections using PicoPureRNA reagents according to Molecular Devices' instructions
|
| Label |
Cy5
|
| Label protocol |
Custom automated version of the aminoallyl MessageAmp II kit from Ambion. 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.
|
| |
|
| |
| 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 |
Individual LCM stroma from esophageal disease
|
| 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.
The raw data files were generated by MATLAB scripts that extracted the data from Rosetta Resolver data files.
The field definitions in the data files are as follows:
ID_REF = Rosetta-generated unique Reporter ID for GPL4380
DETRENDED_RATIO = Detrended, Mean-normalized Log10 Ratio
NORMALIZED_RATIO = Mean-normalized Log10 Ratio
RATIO = Corrected Log10 Ratio of channel intensities (CH2/CH1)
LOGINTENSITY = Corrected average log intensity of channels
INTENSITY1 = Cy3 intensity (CH1)
INTENSITY2 = Cy5 intensity (CH2)
PVALUE = P-value of RATIO
QUALITY = 1 - if good and non control, 0 - otherwise
|
| |
|
| Submission date |
Dec 23, 2009 |
| Last update date |
Jan 22, 2010 |
| Contact name |
James Hardwick |
| E-mail(s) |
james_hardwick@merck.com
|
| Phone |
267-305-4075
|
| Organization name |
Merck & Co., Inc.
|
| Department |
Molecular Profiling
|
| Street address |
351 N. Sumneytown Pike
|
| City |
North Wales |
| State/province |
PA |
| ZIP/Postal code |
19454 |
| Country |
USA |
| |
|
| Platform ID |
GPL4372 |
| Series (1) |
| GSE19632 |
Stromal gene expression in human esophageal cancer |
|
