Array CGH Fresh DNA

DNA extracted from fresh frozen tumors or cell lines is measured by fluorometry and then random prime labeled for CGH arrays.

1) Random Prime Labeling

For each Test sample labeled with Cy3, a corresponding Reference (gender matched) is labeled with Cy-5. Avoid exposure to light as much as possible for the cy dyes.

· The following is prepared in separate tubes

1) Test: 2) Reference

500 ng of Test DNA 500 ng of Reference DNA (normal control)

32 ml 2.5x RP mix 32 ml 2.5x RP mix (Bioprime Kit, Invitrogen)

xx ml of ddH₂0 xx ml of ddH₂0

64.4 ml final reaction volume 64.4 ml

• Vortex, quickspin. Heat to 100°C for 10 min. Use thin-walled PCR tubes. Place on ice immediately for 10 minutes.

• Remove from ice, quick spin then add

8 ml RPL dNTPs (see below)

1.6 ml Klenow enzyme (from Invitrogen BioPrime Kit)

6 ml cy3-dUTP (test) or cy5-dUTP (reference) (Amersham/Pharmacia)

15.6 ml total volume

• Vortex gently, quickspin. Incubate for 2-4 hours at 37°C. The longer time allows for more incorporation of the dyes, thus brighter signals.

RPL dNTPS: 4 ml 100 mM dATP, 4 ml 100 mM dCTP, 4 ml 100 mM dGTP

2 ml 100 mM dTTP

2 ml 1 M Tris pH 8 (PCR room)

0.4 ml 0.5 M EDTA (PCR room)

183.6 ml dH₂O

200 ml total

4) Sephadex column purification of labeled probe: Purify in Amersham G50 Column

• mix column by vortexing, snap off bottom, loosen cap, place column in 1.5 ml screw cap tall tube, spin 1 min at 735x g.
• Place column in new tube, spin 2 mins at 735 x g. Labeled probe will be in tube. You should be able to see a pinkish tint for the test and a bluish tint for the reference. If it is difficult to see the color when holding the tube over a white piece of paper then the samples probably did not label efficiently. Samples may be stored at -20°C protected from light.

Array CGH Hybridization

DAY 1:

1) Reprecipitate DNA's :

Avoid exposure to light as much as possible for the cy dyes.

• Add the following DNAs to a 1.5 ml centrifuge tube, mixing with pipet:
  100 ug Human Cot-1 DNA (100 ml of 1mg/ml Invitrogen cot DNA)
  ~500 ng Cy3 labeled test DNA (~80 ml, which is the volume after the sephadex column of the fresh DNA labeling)
  ~500 ng Cy5 labeled reference DNA (~80 ml, which is the volume after the sephadex column the fresh DNA labeling)
• Add 1/10th volume of room temperature 3M Na Acetate, mixing with pipet.
• Add 2.5 X (original) vol 100% EtOH to ppt DNA, vortex gently.
• Spin 30 mins at 14K rpm (20K x g), 4 °C.

NOTE: while probes are spinning you can begin preparing the slides as in step 2. Slides can also be prepared before you precipitate the probe mixes.

• Decant supernatant; blot dry, being careful to avoid DNA pellet. Allow pellet to dry for about 10 minutes.
• Carefully add a 18 ml mixture containing 6 ml yeast tRNA (sigma, 100 ug/ml); 8 ml 20% SDS; 4 ml dH20. Do not resuspend with pipet, allow the pellet to slowly redissolve in the mixture for 5 minutes, then vortex vigorously, quick spin down, let sit for another 5 minutes, vortex vigorously, quick spin down. It is critical that the pellet is resuspended well before the next step.
• Add 42 ml of room temperature MM 1.0, do not resuspend with pipet, vortex vigorously, quick spin down, let sit for another 5 minutes, vortex vigorously, quick spin down. If everything is dissolved then you can continue. It is ok to leave it longer to ensure full resupension of the probe.
• Quickly spin (1 sec) to bring the probe mix to bottom of tube.

2) Slide Preparation:

NOTE: while probes are reprecipitating as above, you can begin preparing slides. Slides can also be prepared before you precipitate the probe mixes

• Select slides with arrays that have no marks on the chromium or missing chromium. Array spots should all be nice and round and not touching.
• UV cross link the slide at 1200 mjoules in a stratalinker (Stratagene)
• Mark the array carefully on the top edges with a minimal mark using a diamond pencil (about 2 mm away from outer edges of array). You do not want to etch too deeply or into where the rubber cement border is going to be. Remove any pen marks from the slide, and etch the number on the label side so it is clear which is the top of the slide.
• Using a 22 Gauge Needle and a 3 cc syringe carefully make a rubber cement dam around the two arrays. Make sure to leave at least 2 mm from the edge of the array and the rubber cement border. Allow rubber cement to dry at RT for about 10 minutes or more. Protect arrays from dust.

• Place slide on 37 °C slide warmer for a minimum of 30 minutes before adding the probe mix.

3) Hybridization:

• Place slides on 37 °C warmer 30 minutes before adding probes.
• Denature the resuspended probe mix at 70 °C for 10-15 minutes.
• Transfer immediately to a 37 °C water bath and incubate for 30 minutes. (probes can be at 37 °C a little longer if necessary due to handling multiple samples at once. It is best to time the denaturation and 37 °C incubation for the probes in sets of 4 (for two slides).
• Prepare slide incubation chamber. We use a flat rectangular plastic slide holder with the opening on one end. We place a rubber cement dam on the bottom of the plastic container and they can be reused indefinitely. Add 300ul of liquid (we use the HWB wash buffer) in the bottom of the chamber and place chamber on the 37 °C warmer.
• Quick spin down probe mix, resuspend gently with pipet, then apply warm probe immediately onto warmed slide on the hot plate. Spread out the probe mix while adding, and avoid bubbles as much as possible. Repeat for second array, then immediately rock the slide back and forth to spread out the probe mix over the whole array, making sure it reaches all edges of the rubber cement. Sometimes it needs a little help and you can use a pipet tip to gently coach it to the edge.
• Apply probes to second slide, and repeat the spreading out technique, leaving first one on the 37°C warmer. Bubbles from the SDS should disperse.
• It is critical that the probe mix is well dispersed, has minimal bubbles before placing it into the slide chamber. Place slides into chamber taking care to keep the slides level, and to avoid disrupting the rubber cement. Seal the chamber with a layer of rubber cement, then a layer of parafilm. Keep the bottom of chamber as flat as possible, do not fold the parafilm on the bottom.

• Incubate for 2 days at 37 °C in a humid chamber on a slowly rocking platform. Move the chamber to the other end of the rocker 2 times per day to endure equal mixing.

DAY 2: WASHES AND STAINING

• Remove slide from chamber keeping it level, and carefully remove the rubber cement, make sure the array is remaining, moist. If it is starting to dry out then stop and place it into the washes, you can remove the remainder while in the PN step.
• Rinse the slide once with PN, then place into pre-warmed HWB (50% formamide/2XSSC) at 48 °C. Swish slide and then incubate for 15 minutes.
• Wash 1X for 20-30 minutes at 48°C in 2X SSC/ 0.1% SDS. (the longer time is for when there are more than 2 slides). Do not wash more than 4-5 slides at a time.
• Wash 1X for 10 mins at RT in PN, remove any remaining rubber cement during this step, taking care to keep slide fully wet.
• Wash 1X for 10 mins at RT in PN.
• Remove slide from PN, blot back and one side, and leave slide wet, then add 100 ml of RT 0.3ug/ml DAPI in PBS buffered glycerol, and carefully place a 22x50 mm coverslip over arrays, avoiding bubbles. Blot out excess DAPI and PN from the edges onto kimwipes and carefully seal the edges with clear nail enamel.

DAY 3: IMAGING

• Slides are usually left flat, overnight at RT in dark container and imaged the following day.
• Chromium slides must be imaged using a CCD camera set up, typical exposure times on our current in house system, are: DAPI 2000ms; Cy3 45-60,000 ms; cy5 180,000 ms. Clear slides can be imaged on the axon scanner, with typical pmts being for cy3 540, and for cy5 680

NOTES ON array CGH VARIABLES:

Cot-1 DNA: Cot DNA varies from lot to lot, and we measure concentration using fluorometry. Greater than 500 ng/ml is acceptable (1 mg/ml is the expected concentration)

Probe DNA: The amount of probe DNA used can be varied depending on the size of the array. We use all the probe from one reaction, and dissolve it in 60 ml for an array that measures 12x12mm.

Slides: The quality of the slides used is important and should be checked for debris on the slides, smudges, and that the targets are nice and round and not touching each other.

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S. DeVries / F. Waldman

Email questions to Sandy DeVries at the UCSF Cancer Center (devries@cc.ucsf.edu)

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