|
| Status |
Public on Apr 10, 2010 |
| Title |
epithelial cells (PC-A5) |
| Sample type |
RNA |
| |
|
| Source name |
Epithelial cells, captured by LCM
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: epithelial cells
disease state: Prostatic adenocarcinoma
gleason score: Gleason (3+3)
age(yrs): 62
other: same tumor as D9
|
| Biomaterial provider |
The serial frozen tissue sections for LCM were provided by The Ohio State University Prostate Cancer tissue Bank, part of the Human Tissue Resource Network (HTRN) in the Department of Pathology (Columbus, Ohio)
|
| Treatment protocol |
Frozen sections were stained and dehydrated as recommended by manufacturer of the HistoGene LCM staining kit (Arcturus Bioscience, Molecular Devices). The slide was dehydrated through increasing concentrations of Ethanol and Xylene then placed in the Laser Capture Microscope (Arcturus Auto Pix). Areas of the slide with the most abundant glandular epithelial cells were identified and a Cap-Sure HS LCM cap (Molecular Devices) was aligned over the tissue. Microscopy was performed to verify cells were removed from sections and retained on the cap. After isolating the epithelial cells then the same marked slide was aligned for additional collection of interstitial stromal cells . Cell capture and lysis was completed within 2 hours to maintain quality RNA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cells captured by the LCM were lysed and RNA extracted as per manufacturer’s recommendations for Pico Pure RNA isolation kit (Arcturus Bioscience, Molecular Devices). After treatment with DNase, the RNA quantity and quality were checked using the Agilent RNA Pico-Chip on the Bioanalyzer 2100 (Agilent Bioscience, Mt View, CA). RNA quality was measured by 18s/28s ratio and degraded RNA samples were omitted. 1 ng of RNA was amplified using the RiboAmp HS kit (Arcturus Bioscience, Molecular Devices) in two-rounds of linear amplification of RNA.
|
| Label |
biotin
|
| Label protocol |
The total amplified cDNA was biotin labeled as recommended by the Affymetrix GeneChip Expression Analysis Technical Manual (2004).
|
| |
|
| Hybridization protocol |
Following fragmentation, 10 ug of biotin labeled cRNA was hybridized for 16 hr at 45C to Affymetrix Human Genome U133A 2.0 chips (HG_U133A 2.0) according to the Affymetrix GeneChip Expression Analysis Technical Manual (2004). The hybridized arrays were washed and stained in the Affymetrix Fluidics Station 400 using the Midi_euk2v3 labeling kit for detection by scanning.
|
| Scan protocol |
The U133A 2.0 chips were scanned using an Affymetrix® GeneChip® scanner 3000. The signal intensities were normalized to the spike-controls located on the array chip. Platform used was GPL571.
|
| Description |
Gene Expression data from LCM captured prostate cancer epithelial cells (PC-A5), same tumor as D9.
|
| Data processing |
The data were analyzed with GeneChip Operating System 1.1 using the MAS 5.0 algorithm, with Affymetrix default analysis settings and global scaling as normalization method, and using the default normalization target setting of 500.
|
| |
|
| Submission date |
Mar 11, 2010 |
| Last update date |
Mar 11, 2010 |
| Contact name |
Gail C Fraizer |
| E-mail(s) |
gfraizer@kent.edu
|
| Phone |
330-671-1398
|
| Fax |
330-672-3713
|
| Organization name |
Kent State University
|
| Department |
Biological Sciences
|
| Street address |
251 Cunningham Hall
|
| City |
kent |
| State/province |
OH |
| ZIP/Postal code |
44242 |
| Country |
USA |
| |
|
| Platform ID |
GPL571 |
| Series (1) |
| GSE20758 |
Expression data from LCM captured prostate cancer cells |
|
