|
| Status |
Public on Feb 23, 2007 |
| Title |
Joosse 2007: SKBR3 - manual hybridization |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
SKBR3 cell-line
|
| Organism |
Homo sapiens |
| Characteristics |
organ: mammary gland; breast
disease: adenocarcinoma
derived from metastatic site: pleural effusion
age: 43 years
gender: female
ethnicity: Caucasian
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were suspended in 1 ml DNAzol reagent, centrifuged at 14000rpm for 10 min at 4C. The supernatant was transferred to a new tube and 0.5 ethanol absolute was added to precipitate the DNA with swirling. The DNA precipitate was transferred to a new tube and washed with 1 ml 75% ethanol. The ethanol was removed and the DNA was disolved in 50ul TE buffer.
|
| Label |
Cy5
|
| Label protocol |
According to the manufacturers’ instructions (Kreatech Biotechnology, Amsterdam, http://www.kreatech.com/).
|
| |
|
| Channel 2 |
| Source name |
Women reference pool
|
| Organism |
Homo sapiens |
| Characteristics |
A pool of DNA from peripheral blood lymphocytes, from 7 apperently healty women.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Equipment and reagents
Lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 1 mM EDTA), DNAzol (Invitrogen), TE buffer pH 8.0, 10 mM Tris.HCl, 1 mM EDTA
Lysis
1. Mix the blood carefully and decant in a 50 ml tube
2. Rinse the EDTA tube with lysis buffer and decant this together with the blood
3. Fill the tube to 50 ml with lysis buffer and mix
4. Put the tube on ice for 10 minutes
5. Centrifuge at 3000 rpm for 10 minutes at 4°C
6. Poor off the supernatant
7. Carefully resuspent cell pellet in 50 ml lysis buffer
8. Centrifuge at 3000 rpm for 10 min at 4°C
9. Poor off supernatant
10. Repeat wash steps until the supernatant is a clear solution
11. Sample can be stored in liquid nitrogen or at -70°C
Genomic DNA isolation
1. Add 1 ml DNAzol to the pellet and mix by pipeting until clear solution is left
2. Add 500 ml 100% EtOH and mix carefully
3. Transfer the DNA pellet with a sterile öse to tube with 1 ml 70% EtOH
4. Wash the DNA and press as many EtOH out of the pellet as possible
5. Transfer the DNA to a 2 ml Eppendorf tube with 1 ml TE buffer
6. Solute by rotating overnight at 37°C
|
| Label |
Cy3
|
| Label protocol |
According to the manufacturers’ instructions (Kreatech Biotechnology, Amsterdam, http://www.kreatech.com/).
|
| |
|
| |
| Hybridization protocol |
Hybridization as described in: Van Beers EH, Joosse SA, Ligtenberg MJ, Fles R, Hogervorst FBL, Verhoef S, Nederlof PM: A multiplex PCR predictor for aCGH success of FFPE samples. Br J Cancer. 2006 Jan;94(2):333-7
|
| Scan protocol |
Slides were scanned with an Agilent DNA Microarray Scanner BA on the same day.
|
| Description |
-
|
| Data processing |
Data processing included signal intensity measurement in ImaGene Software and median pintip (c.q. subarray) normalization.
|
| |
|
| Submission date |
Feb 21, 2007 |
| Last update date |
Jun 10, 2009 |
| Contact name |
Simon A Joosse |
| E-mail(s) |
[email protected]
|
| Organization name |
University Medical Center Hamburg-Eppendorf
|
| Department |
Institute of Tumor Biology
|
| Street address |
Martinistrasse 52
|
| City |
Hamburg |
| ZIP/Postal code |
20246 |
| Country |
Germany |
| |
|
| Platform ID |
GPL4560 |
| Series (1) |
| GSE7122 |
Automated array-CGH optimized for archival formalin-fixed, paraffin-embedded tumor material |
|
