|
| Status |
Public on Nov 01, 2012 |
| Title |
genomic DNA from Panc02.03 cell line (copy number) |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Panc02.03
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Panc02.03
gender: female
disease state: pancreatic adenocarcinoma
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted by the standard phenol-chloroform extraction followed by ethanol precipitation
|
| 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 |
sample type: anonymous male diploid reference DNA
disease state: normal
gender: male
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted by the standard phenol-chloroform extraction followed by ethanol precipitation
|
| 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 |
Mix the two reactions (reference and sample) and add 100ul Cot-1 DNA (1ug/ul), and purified using a microcon YM-30 column. Resuspend the probe in 45 uL hybridization solution (36 uL DIGeasy+ 4.5 uL (20mg/mL) sheared Herring Sperm DNA + 4.5 uL (10mg/mL) yeast tRNA). Denature the samples at 85°C for 5min, and incubate at 45°C for 1 hour to allow Cot-1 to anneal to DNA probe. Add 45uL of the probe solution onto the array slide. Gently lower coverslip (22x60mm, Fisher Scientific) onto slide over probe solution. Place the slide into a pre-warmed (45°C) hybridization cassette (Telechem) and add 10uL of H20 in the lower groove (to control humidity). Incubate for 36 - 40hrs at 45°C. Wash the slide with 0.1X SSC + 0.1% SDS (pH 7.0, pre-warmed to 45°C) for 5 minutes in the dark with agitation, and with 0.1X SCC 5 times. Dry the slides by centrifuging at 500rcf for 1 minute.
|
| Scan protocol |
Hybridizations were scanned using the Axon GenePix 4000B scanner. Array images are analyzed with GenePix Pro 6.1.
|
| Data processing |
A three-step normalization procedure, including LOWESS fitting, spatial, and median normalization was used.
|
| |
|
| Submission date |
Oct 23, 2012 |
| Last update date |
Nov 01, 2012 |
| Contact name |
Kelsie L Thu |
| Organization name |
BC Cancer Research Centre
|
| Street address |
675 West 10th Avenue
|
| City |
Vancouver |
| State/province |
British Columbia |
| ZIP/Postal code |
V5Z 1L3 |
| Country |
Canada |
| |
|
| Platform ID |
GPL8774 |
| Series (2) |
| GSE40099 |
Pancreatic cancer and non-malignant pancreas cell lines |
| GSE41794 |
DNA copy number profiling of 20 pancreatic cancer cell lines |
|
