|
| Status |
Public on Dec 13, 2011 |
| Title |
3010-lcm |
| Sample type |
RNA |
| |
|
| Source name |
EE_IB_1_cauc_lcm
|
| Organism |
Homo sapiens |
| Characteristics |
race: caucasian
age (years): 58
stage: IB
grade: 1
histology: endometrioid
|
| Treatment protocol |
Laser Capture microscopy was used to dissect cancer cell from tumors or epithelium from normal endometria to provide material for RNA extractions. Approximately 5-8 slides (with tissue samples 10 microns thick) of LCM microdissection were required for each cancer case and 20 slides of LCM were required for each normal. Cancer samples were microdissected to provide >95% purity of cancer. Normal endometria were dissected to provide >95% purity of normal epithelial cells.
|
| Growth protocol |
Fresh frozen cancer specimens from 91 stage I endometrial patients were examined. Normal endometrial samples from twelve age matched postmenopausal women were used for comparison. All tissues were collected under an IRB approved protocol at the corresponding institutions. Specimens were harvested by pathologists and frozen within one hour of excision and each uterine tumor was frozen until the time of analysis. Tissue specimens were evaluated by one of two board certified gynecologic pathologists using H&E staining to confirm the diagnosis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Approximately 50ng of Total RNA was extracted from each sample with the Qiagen RNeasy® Micro Kit (Qiagen, 2003) by the manufacters suggested protocol for Total RNA Isolation from Microdissected Cryosections. See Rneasy® Micro Handbook Qiagen 4/2003.
|
| Label |
biotin
|
| Label protocol |
Extracted RNA was processed using the GeneChip two cycle cDNA synthesis kit (Affymetrix), see Chapter 2 of Gene Chip® Expression Technical Manual (2005-2009 Affymetrix, Inc.) for protocol.
|
| |
|
| Hybridization protocol |
Labeled RNAs were hybridized according to manufacterer's specifications (Affymetrix) for the HG-U133 plus 2.0 Gene Chip system, see Chapter 3 of Gene Chip® Expression Technical Manual (2005-2009 Affymetrix, Inc) for protocol.
|
| Scan protocol |
Hybridized samples were washed, stained and scanned according to manufacterer's specifications (Affymetrix) for the HG-U133 plus 2.0 Gene Chip system, see protocol in GeneChip® Expression Wash, Stain and Scan User Manual.
|
| Description |
End Ca_frozen_LCM
|
| Data processing |
All expression data were normalized to a target intensity of 500 using Affymetrix's MAS5.0 algorithm. Affymetrix .CEL files were processed with the program GCOS to generate signal valuses. All .CEL files were scaled to a global median intensity target intensity of 500. Hieracrchical clustering of samples was performed with Cluster 3.0 and the dendograms were visualized with Tree View (Eisen Lab, University of California, Berkeley). Clustering was performed with the entire gene list (unsupervised) to determine wheter there are groupings of samples and the same software was used to generate heat maps of the most differentially expressed genes. Differential gene expression betweeen groups of samples was performed using BRB array tools from the National Cancer Institute (http://linus.nci.nih.gov/BRB-ArrayTools.html). The biological annotation of differentially expressed gene lists were carried out by a number of methods including DAVID (http://david.abcc.ncifcrf.gov/), IPA (Ingenuity Systems, Redwood City, CA) and MetaCore (GeneGo, San Diego, CA).
|
| |
|
| Submission date |
Jul 09, 2009 |
| Last update date |
Dec 13, 2011 |
| Contact name |
Uma Chandran |
| E-mail(s) |
[email protected]
|
| Phone |
412-648-9326
|
| Organization name |
University of Pittsburgh
|
| Department |
BioMedical Informatics
|
| Street address |
5150 Center Ave
|
| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15232 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE17025 |
Gene Expression Analysis of Stage I Endometrial Cancers |
|
