organ: mammary gland; breast disease: infiltrating ductal carcinoma
Extracted molecule
genomic DNA
Extraction protocol
DNA is extracted 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
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.
