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/).
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
Step 1: Wash at 37°C, 2x SSC (pH 7), 1 wash run for 30 sec, no soak Step 2: Probe injection (pre-hyb mix) at 37°C Step 3: Hybridisation at 37°C for 01:00:00, Agitation Frequency: high Step 4: Wash at 37°C, 2x SSC (pH 7), 1 run, wash time 15 sec, no soak Step 5: Probe injection (sample) at 37°C Step 6: Hybridisation at 37°C for 69:00:00, Agitation Frequency: high Step 7: Wash at 37°C, 2x SSC, 0.1% SDS, 12 runs, wash time 1 min, soak time 1 min Step 8: Wash at 68°C, 2x SSC, 0.1% SDS, 6 runs, wash time 1 min, soak time 1 min Step 9: Wash at 68°C, 2x SSC (pH 7), 2 runs, wash time 1:30 min, soak time 1 min Step 10: Wash at 23°C, 0.1x SSC, 1 wash run for 1:30 sec, no soak Step 11: Slide drying step at 30°C for 2 min, final manifold cleaning using N2 gas
Scan protocol
Slides were scanned with an Agilent DNA Microarray Scanner BA on the same day.
Description
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Data processing
Data processing included signal intensity measurement in ImaGene Software and median pintip (c.q. subarray) normalization.
