|
| Status |
Public on Sep 28, 2012 |
| Title |
Tissue Biopsy of Intermediate Risk Prostate Cancer Patient 16 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Biopsy Tumor Tissue
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Prostate cancer biopsy
relapse rate (bf=biochemical failure, ned = no evidence of disease): NED
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted by the standard phenol-chloroform extraction protocol.
|
| Label |
Cy3
|
| Label protocol |
Random-primer labelling reaction 1. For each slide combine: (1 reaction tube for reference & 1 reaction tube for sample) a) DNA (200ng) b) 5uL of 5X (PB) random primers buffer (Final concentration: 5X Promega Klenow buffer and 7ug/ul random octomers) c) Dilute to 16.5uL total volume with ddH2O 2. Boil for 10 min at 100°C in PCR machine. Transfer immediately to ice for 2 min 3. Add 4ul of 10X dNTP mix (2mM each dATP, dGTP, dTTP, 1.2mM dCTP) - Vortex very well 4. Add CyeDyes: Vortex and spin prior to adding • Add 2uL (2nmoles) of Cy3 labelled dCTP to Reference DNA (Novagen Male Genomic) • Add 2uL (2nmoles) of Cy5 labelled dCTP to sample DNA 5. Add 2.5uL of Klenow (9U/uL) and mix by pipetting 6. Incubate 37oC overnight (~18hours).
|
| |
|
| Channel 2 |
| Source name |
Normal male
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Pooled male genomic DNA [Novegen]
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted by the standard phenol-chloroform extraction protocol.
|
| Label |
Cy5
|
| Label protocol |
Random-primer labelling reaction 1. For each slide combine: (1 reaction tube for reference & 1 reaction tube for sample) a) DNA (200ng) b) 5uL of 5X (PB) random primers buffer (Final concentration: 5X Promega Klenow buffer and 7ug/ul random octomers) c) Dilute to 16.5uL total volume with ddH2O 2. Boil for 10 min at 100°C in PCR machine. Transfer immediately to ice for 2 min 3. Add 4ul of 10X dNTP mix (2mM each dATP, dGTP, dTTP, 1.2mM dCTP) - Vortex very well 4. Add CyeDyes: Vortex and spin prior to adding • Add 2uL (2nmoles) of Cy3 labelled dCTP to Reference DNA (Novagen Male Genomic) • Add 2uL (2nmoles) of Cy5 labelled dCTP to sample DNA 5. Add 2.5uL of Klenow (9U/uL) and mix by pipetting 6. Incubate 37oC overnight (~18hours).
|
| |
|
| |
| Hybridization protocol |
300 ng of sample and reference DNA from pre-treatment prostate biopsies were differentially labeled in a random priming reaction with Cyanine 3–dCTP and Cyanine 5–dCTP (Perkin Elmer Life Sciences). DNA samples were then combined and mixed with 100 μg of human Cot-1 DNA (Invitrogen) followed by removal of unincorporated nucleotides using microcon YM-30 columns (Millipore). Sample mixtures were denatured at 85°C for 10 min, and repetitive sequences were blocked at 45°C for 1 hr before hybridization. The mixture was then applied onto arrays containing 26,819 bacterial artificial chromosome (BAC) derived amplified fragment pools spotted in duplicate on aldehyde coated glass slides (SMRT v.2, BC Cancer Research Centre Array Facility). Array hybridization was performed in the dark at 45°C for 36 hr inside a hybridization chamber, rinsed four times in 0.1X SSC at room temperature and dried by centrifugation at 1,000g before imaging.
|
| Scan protocol |
Slides were scanned using a dual laser array scanner (Axon) and spot signal intensities determined using the SoftWoRx Tracker Spot Analysis software (Applied Precision).
|
| Description |
SAMPLE 16
|
| Data processing |
Normalized using algorithm by Khojasteh et al (PMID: 16297240) : A three-step normalization procedure, including LOWESS fitting, spatial, and median normalization.
|
| |
|
| Submission date |
Sep 26, 2012 |
| Last update date |
Nov 14, 2014 |
| Contact name |
Igor Jurisica |
| Organization name |
Princess Margaret Cancer Center
|
| Department |
TMDT 11-413
|
| Street address |
101 College St.
|
| City |
Toronto |
| State/province |
ON |
| ZIP/Postal code |
M5G1L7 |
| Country |
Canada |
| |
|
| Platform ID |
GPL2616 |
| Series (1) |
| GSE41120 |
Molecular Characterization of 126 Intermediate Risk Prostate Cancer Patient Samples |
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