Microarray Core UCSF Comprehensive Cancer Center Standard Operating Procedure Title: Array CGH Hybridization Protocol HumArray 3.2 SOP No.: MC023QA Version: 5 Date: 5-12-06 Page No.: 1 of 5 Authors: Albertson, Pinkel, Blackwood, Segraves, Snijders, Huey. Contents; Overview 1.0 Materials 2.0 Methods 3.0 Notes 4.0 Acknowledgements & References Reviewed: Hamilton Overview This procedure explains the steps to hybridize differentially labeled human probe DNA samples to our human BAC array printed on a chromium coated glass slide. These procedures have proven to be reliable in our laboratory. 1.0 Materials Hybridization of fluorescently labeled genomic DNA for array CGH analysis 1. Differentially labeled test and reference genomic DNA from MC 020. 2. Human Cot-1 DNA (1 mg/ml, Invitrogen) (see Note 2). 3. 20% SDS in sterile H 2 O (heat to 68 C to dissolve). Store at RT. 4. 100% Ethanol. Store at -20 C. 5. 3.0 M Sodium acetate ph 5.2. 6. Dextran sulfate (sodium salt, 500,000 MW). 7. Formamide (re-distilled, ultra pure, Invitrogen). Store at -20 C. 8. 20x SSC (3.0 M NaCl, 0.3 M sodium citrate, ph 7.0). 9. Master Mix mixture: dissolve 1 g dextran sulfate (sodium salt, 500,000 MW, available from Fisher Biotech) in 5 ml of formamide (re-distilled, ultra pure) and 1 ml of 20x SSC (see Note 3). Bring volume to 7mL with dh2o. Bring to ph 7.0 with approximately 2 drops of HCl. 10. PN buffer: 0.1 M sodium phosphate, 0.1 % nonidet P40, ph 8.0 (see Note 4). 11. Sterile H 2 O (e.g. autoclaved, deionized and filtered water).
12. UV Stratalinker 2400 (available from Stratagene) capable of producing 130,000 x 100μJoules UV. 13. Very slow rocking table (~ 1 rpm) inside a 37 C incubator (e.g. a VWR brand Rocker, Model 100). 14. Rubber cement (Ross, American Glue Corporation). 15. Silicone gasket (Press-to-Seal, 2mm thick, #62-6508-24, PGC Scientific). 16. 100% Glycerol. 17. 10x PBS: 1.4 M NaCl, 0.027 M KCl, 0.1 M Na 2 HPO 4, 0.018 M KH 2 PO 4, adjusted to ph 7.4 with HCl. 18. Stereomicroscope. 19. 10 ml Syringe. 20. 200 μl Disposable pipet tip without a filter. 21. Binder clips, medium size. 2.0 Methods Hybridization of fluorescently labeled genomic DNA for array CGH analysis 1. Preparation of array for hybridization: a. Expose a printed array to 260,000 μjoules (2,600 x 100uJoules) of UV by using a Stratalinker (see Note 5). b. Fill a 10 ml syringe with rubber cement and fit a 200 μl pipet tip on the syringe outlet. You may have to cut 1-2mm off the wide end of the pipet tip for it to fit well. Apply a rubber cement ring around each array on the slide using a stereomicroscope to observe the area of the array. Air-dry the rubber cement and apply a second thick layer of rubber cement on top of the first layer. Air-dry the rubber cement. 2. Preparation of samples for hybridization: a. Combine 30 μl labeled test genomic DNA, 30 μl labeled reference genomic DNA, (see MC 022QA) and 75 μg of human Cot-1 DNA. The volume of Cot1 will depend on the Cot1 concentration. Precipitate the DNA sample mixture by adding 2.5 volumes of ice-cold 100% ethanol and 0.1 volume of 3 M sodium acetate (ph 5.2). Vortex the solution briefly but
thoroughly and collect the precipitate by centrifugation at 14,000 rpm for 45 minutes at 4 C. b. Carefully aspirate and discard the supernatant. Wipe the excess liquid from the tube with a chem-wipe paper tissue, being careful not to disturb the pellet. Air-dry the pellet for approximately 5-10 minutes. Dissolve the pellet in 7μL dh 2 O, 14μL 20% SDS, and 49 μl Master mix mixture (for Master Mix preparation see Note 3). The pellet may need to incubate at room temperature on the bench for an hour to completely re-suspend. 3. Denature the DNA sample solution from step 2.2.b in a water bath at 73 C for 13 minutes and then incubate at 37 C for 60 to 120 minutes to allow the Cot1 DNA to anneal to repetitive sequences on both the sample and reference DNA. 4. Place Array on a heat block set at 37 C for 5 minutes to warm array. 5. Apply the combined sample/reference hybridization solution onto the array (see Note 7). Keep sample/reference hybridization solution at 37 C until just before application to the array to reduce non-specific binding of the probe to the array surface. Place a silicon gasket around the edge of the slide and lay a clean glass slide on top, aligning the edges with the gasket. Clamp the assembly together using binder clips. Incubate the array for 48 to 68 hours at 37 C on a slowly rocking table (~1rpm). 6. Disassemble the slide assembly and rinse the hybridization solution from the slide under a stream of PN buffer. It is preferred to leave the rubber cement on the array at this time, as it will not affect the rinsing steps that follow. 7. Wash the slides once in 50% formamide, 2x SSC, ph7 for 15 minutes at 45 C, followed by a 15 minute wash in PN buffer at room temperature. The washes can conveniently be done in slide staining jars (coplin jars) placed in water baths. 8. At the bench, carefully remove the rubber cement with forceps, while keeping the array moist with PN buffer. 9. Mount the slide in a DAPI solution to stain the array spots (90% glycerol, 10% PBS, 1 μm DAPI). 10. Arrays are ready for imaging (see Note 7).
3.0 Notes 1. DNA concentrations should be determined using a Fluorometer, rather than a spectrophotometer to obtain more accurate measurements. 2. The DNA concentration of each new lot of Cot-1 DNA should be determined using a Fluorometer and should measure 500 ng/μl or greater. 3. Distribute 1 g of dextran sulfate over the entire length of a 15 ml tube. While holding the tube horizontally, squirt in 5 ml of formamide. Close the tube and shake vigorously for 30 seconds. Add 1 ml of 20x SSC and shake vigorously for 30 seconds. Add 1 ml of dh 2 O and mix thoroughly. Bring ph to 7.0 using approximately 2 drops of HCl. Dissolve overnight at room temperature. The final volume should be approximately 7 ml. 4. Prepare approximately 16 L of 0.1 M Na 2 HPO 4, 0.1% nonidet P40 ph9. While continuously measuring the ph, adjust the ph to 8.0 with 0.1 M NaH 2 PO 4, 0.1 % nonidet P40 (approximately 1 L). Make sure not to go below ph 8.0. Store at room temperature. 5. Place the slide(s) in the Stratalinker, arrays facing up. The arrays should be given a fixed amount of energy (260,000 μj or 2,600 x 100 μj) instead of other available options the Stratalinker might have, such as autocrosslink or time. Overcrosslinking the slide might result in a decrease in fluorescent hybridization signal. 6. Place about 10 μl of the hybridization solution in each corner of the array, and the remaining 30 μl in the center. Tilt slide until entire surface of array is wet with hybridization solution. Remove any air bubbles with needle or other sharp object. 7. Images can be acquired using commercially available CCD or laser scanning imaging systems (e.g. an Axon scanner 4000B). Image analysis and quantification can be done using commercially available image analysis programs (e.g. GenePix from Axon) or the UCSF program Spot. 4.0 Acknowledgements & References 1. R. Segraves, A. Snijders, and B. Huey were instrumental in preparing and proof reading this protocol.
2. Snijders, A., Segraves, R., Blackwood, S., Pinkel, D., Albertson, D., (2001) BAC Microarray-based Comparative Genomic Hybridization, Comprehensive Cancer Center, Cancer Research Institute and Department of Laboratory Medicine, UCSF, San Francisco.