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ISO 9001:2000

BAC aCGH labeling and hybridization protocol v821.7

The following established procedure details a direct labeling of 1ug of genomic DNA using a Bioarray kit from Enzo Life Sciences, and subsequent hybridization to a BAC arrayed slide using either a Genetac Hybridization station or an Agilent Rotisserie Hyb Oven. Perform competitive hybridizations pairing the experimental sample (labeled with cy5 dCTP) to a pooled control of the opposite gender (labeled with cy3 dCTP).

Materials

  • Bioarray CGH labeling system (Enzo, Cat #42670)
  • Qiaquick PCR Cleanup Kit (Qiagen, Cat #28106)
  • Human or Mouse Cot –1 (Invitrogen, Cat #s 15279-011 and 18440-016 respectively)
  • Yeast tRNA (Invitrogen, Cat #15401-011)
  • Slide Hyb # 3 buffer (Ambion, Cat #8863G)
  • 20X SSC (Invitrogen, Cat #15557-044)
  • 10% SDS (Invitrogen, Cat#24730-020)
  • 100% ETOH
  • PCR strip tubes
  • Thermocycler
  • Water bath
  • Slide carrier and staining dishes

For Rotisserie Hybs only:

  • Gasket slides (Agilent, Cat #G2534-60003)
  • Hybridization chamber (Agilent, Cat#G2534A)
  • Hybridization oven (Agilent, Cat#G2545A)

Prepare the following wash solutions:

  • 1X SSC, 0.1% SDS (500 ml per one day of experiments)
  • 0.1X SSC, 0.1% SDS (1000 ml per one day of experiments)
  • 0.1X SSC (1000 ml per one day of experiments)

Yeast tRNA resuspension:

  • Add 250 ul of ddH2O to 1 vial of 25 mg of yeast tRNA to get a 100 ug/ul concentration

Labeling Procedure (Day 1)

  1. Label appropriate number of thermocycler tubes (2 for each experiment) and place on ice
  2. Add 40 ul of Random Primer (#1) to each tube
  3. Add 1 ug of test DNA and add 1 ug of pooled control DNA to their respective tubes
  4. Add water (W) to each tube to bring the total volume up to 89 ul
  5. Cap and gently vortex tubes, place back on ice
  6. Heat denature the tubes on a thermocycler using the following steps:
    • 99C - 10 minutes
    • 4C - 2 minutes (minimum)
  7. Place tubes back on ice
  8. Uncap tubes and add the following in this order:
    • 10 ul of dNTP-cyanine 3 mix (#2) to control DNA, dNTP-cyanine 5 (#3) to test DNA
    • 1 ul of Klenow (#4) to each tube
  9. Cap and gently vortex tubes, place back on ice
  10. Place tubes in a thermocycler or in a water bath at 37 C for 4 hours

Cleanup/Hybridization Procedure (Day 1)

  1. Remove tubes from thermocycler
  2. Add 5 ul of Stop Buffer (#5) to each tube

Qiaquick cleanup

  1. Transfer labeling solutions (approximately 105 ul) from thermocycler tubes to new 1.5 ml Eppendorf tubes
  2. Add 500 ul of PB buffer,vortex, spin down
  3. Place entire volume (approximately 600 ul) onto column
  4. Spin at 13K rpm for 1 minute at room temperature
  5. Remove flow-through, add 750 ul PE Wash buffer
  6. Spin at 13K rpm for 1 minute
  7. Remove flow-through, spin dry column for 1 minute at 13K rpm
  8. Transfer column to a new elution tube, add 25 ul Elution buffer
  9. Let sit for 1 minute, then spin at 13K for 1 minute
  10. Add another 25 ul Elution buffer to the column
  11. Let sit for 1 minute, then spin at 13K for 1 minute

Yield and Incorporation

  1. Gently vortex elutions before reading on the Nanodrop 1000
  2. Open the Nanodrop program, select “Microarray”, initialize with water, select "DNA-50"
  3. Blank the nanodrop with the elution buffer from the cleanup procedure
  4. Measure blank again as a sample; the blank should read no more or less than +/- 1 ng/ul
  5. Measure sample elutions
  6. The samples must meet the following yield and pmol requirements to proceed to hybridization:
    • Concentration (total yield) – 80 ng/ul minimum
    • Cy5 (total pmol of test sample) – 2.6 pmol/ul minimum
    • Cy3 (total pmol of control sample) – 4 pmol/ul minimum
  7. Add cy3 elution (approximately 49 ul) to paired cy5 elution

Ethanol precipitation

  1. Add 100 ul of 1 ug/ul species-specific Cot-1 to cy3/cy5 elution, vortex, spin down
  2. Add 1/10 volume NaAc (approximately 20 ul) and 2.5X volume 100% EtOH (approximately 500 ul) , vortex, spin down
  3. Precipitate at –20 to –80C for a 30 minute minimum. Samples can be stored at -80 C at this point.

Hybridization with Genetac Hybstation

  1. Spin ETOH precipitate at 13K rpm for 15 minutes at 4C
  2. Remove supernatant with pipetman and air dry pellet for 5 minutes
  3. Resuspend pellet in 15 ul ddH2O
  4. Add 5 ul of 100 ug/ul yeast tRNA, vortex, spin down
  5. Heat probe at 95C for 5 minutes
  6. Add 105 ul of Ambion Slide Hyb Buffer #3(prewarmed at 70c), vortex, spin down
  7. Heat probe at 37C for 30 minutes
  8. During 37C incubation, prepare the BAC slide for hybridization. Remove the slide from storage dessicator and place in a UV crosslinker. Set the crosslinker to 350 mJ and crosslink slides.
  9. Place slide on Gentac and begin hybridization protocol, with the following parameters:
    • 37C loading temperature
    • 55C hybridization temperature for 16 hours (agitation enabled)
    • 1st wash
      • 65C (30s wash,30s hold) x 1
      • 55C (30s wash, 30s hold) x 2
    • Add entire probe (approximately 120 ul) to prewarmed (37 C) slide.
    • Hybridize at 55C for 16 hours.

Hybridization with Agilent Rotisserie Hyb Oven

  1. Spin ETOH precipitate at 13K rpm for 15 minutes at 4C
  2. Remove supernatant with pipetman and air dry pellet for 5 minutes
  3. Resuspend pellet in 15 ul ddH2O
  4. Add 5 ul of 100 ug/ul yeast tRNA, vortex, spin down
  5. Heat probe at 95C for 5 minutes
  6. Add 500 ul of Ambion Slide Hyb Buffer #3 (prewarmed at 70C), vortex, spin down
  7. Heat probe at 37C for 30 minutes
  8. During 37C incubation, prepare the BAC slide for hybridization. Remove the slide from storage dessicator and place in a UV crosslinker. Set the crosslinker to 350 mJ and crosslink slides.
  9. After 30 minute incubation, spin down samples for 30 seconds at 13K rpm.
  10. Place a gasket slide in an Agilent hybridization chamber.
  11. Following standard Agilent hyb procedure, load 490 ul of the sample onto the gasket slide in a "drag and dispense" manner.
  12. Place BAC slide "array side" down onto the gasket slide; make sure that the sandwich pair is properly aligned.
  13. Close Hybridization chamber and place the chamber in the Hyb Oven.
  14. Hybridize on 20 rpm setting at 65C for 16 hours.

Post Hyb Wash and Scanning Protocol (Day 2)

Post Hyb Wash

  1. First wash done on Genetac (not done for Rotisserie hybs):
    • 1XSSC, 0.1%SDS
      • 65C (30s wash, 30s hold) x 1
      • 55C (30s wash, 30s hold) x 2
    • For Genetac and Rotisserie Hybs – disassemble hyb chamber in first wash buffer and proceed with the following washes:
      • 0.1X SSC, 0.1% SDS
        • 50 C (30s plunge, 30s dwell) x 2
      • 0.1X SSC
        • 50 C (30s plunge, 30s dwell) x 2
    • Quickly plunge the slide carrier into a room temperature 0.1X SSC-filled staining dish and then a 100% ETOH-filled staining dish (1 second), quickly place slides into 50 ml conical tubes
    • Spin tubes down at 1500 rpm for 3 minutes
    • Store slides in dark until scanned

Scanning

Slides are scanned on the Genepix 4200AL scanner using Genepix 6.1.
Hardware settings will vary per scanner but current laboratory settings are:

  • 635 PMT setting: 400
  • 635 laser power: 50%
  • 532 PMT setting: 400
  • 532 laser power: 20%
  • Pixel size: 5 uM
  • Lines to average: 1
  • Focus position: 0 uM
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