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Comparative genomic hybridization (CGH)
Normal metaphase preparation:
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Collect 5mL whole blood from volunteer and transfer to a centrifuge tube with 500mL ACS. Centrifuge at 14000 rpm for 20mins at room temperature.
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Equally transfer the first and the second layers to three T25 culture flasks in a total volume of 10mL completed medium with 300mL ACS and 100mL PHA.
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Mix the content of the flasks gently.
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Incubate in the humidified incubator with 5% CO2 at 37°C for 3 days. If CO2 incubator is unavailable, use a closed system by capping the tubes tightly.
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Gently mix the content daily.
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Add 50mL 10mg/mL colcemid for 45 minutes at 37°C on the last day of culture.
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Transfer all the content to a 15-mL centrifuge tube. Centrifuge at 1000 rpm for 5 minutes at room temperature. Discard the supernatant and leaving 0.5mL on top of the pellet. Resuspend the pellet well.
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Add 2mL prewarmed (37°C) 0.075M KCl drop by drop and mix the tube gently. Make up to 14mL solution with prewarmed 0.075M KCl and place in 37°C water bath for 15-20 minutes (depending on the species and mitotic inhibitors used). Meanwhile, prepare fresh fixative (1X 100% glacial acetic acid, 3X 100% methanol) and place on ice bath to chill until use.
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Centrifuge at 1000 rpm for 5 minutes at room temperature. Discard the supernatant and leave 0.5mL above the pellet. Be-care not to disrupt the underneath pellet.
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Resuspend the pellet thoroughly. Pipette up all the material with glass pasteur pipette. Add approximately 5mL of cold fixative to the tube. Add the cells to the cold fixative in drop by drop fashion, and mix them well gently. Make up to 10mL with cold fixative. Incubate at -20°C for overnight.
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Centrifuge at 1000 rpm for 5 minutes. Wash the pellet with fresh fixative twice. After the third rinse and spin, leave enough supernatant to make a milky cell suspension and prepare the normal metaphase slide now. Add 8mL fixative to the remaining and store it at -20°C until use.
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Incubate the slides at 37°C for 3 days before use. It is recommended that just prepare the slide when you use it as the slides is not preferred for hybridization if incubate for more than 2 weeks.
DNA probe labelling (Nick Translation labelling):
1. Add the following components to the tube:
(19.5 – x ) mL nuclease-free water
x mL for 1 mg extracted genomic DNA
2.5 mL 0.2mM SpectrumGreen (label test DNA) or
SpectrumRed (label reference DNA) dUTP
5 mL 0.1mM dTTP
10 mL dNTP mix
5 mL 10X Nick Translation buffer
8 mL Nick Translation enzyme
2. Mix the tube briefly.
3. Incubate at 15°C for 2 hours and then temporarily stop the reaction by placing the tube at -20°C.
4. Check the size of the labeled probes by gel electrophoresis, looking for the peak size between 300-800 bp DNA fragments.
5. If the desired probe size is reached, goto step 6; otherwise, continued the incubation at step 4 until adequate size of DNA is obtained.
6. Stop the reaction by heating the probe DNA at 75°C.
7. Store the probe at -20°C until use.
Probe Mix prepararion:
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Mix equal amount of labelled reference and test DNA in a 1.5mL microcentrifuge tube approximately.
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Add 5mL Human Cot-1 DNA and make up to 100mL with dH2O.
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Add 10mL 3M sodium acetate solution into the DNA sample.
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Add 250mL cold absolute ethanol to the mixture and mix well.
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Incubate the mixture at –20°C for at least 30-60 minutes.
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Centrifuge the mixture at 14000rpm at 4°C for 20 minutes.
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Discard the supernatant and vacuum dry the DNA probe.
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Store the DNA probe at –20°C until use.
In situ hybridization:
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After 3 days incubation, pre-treat the normal metaphase slide with 200mL diluted RNase solution (0.1mg/mL in 2X SSC) for about 30 to 60 minutes at 37°C.
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During incubation step 1, prepare the hybridization mixture as follows:
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Dissolve DNA probe in 3mL dH2O together with 7mL MM2.1
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Denature the hybridization mixture at 75°C for 5 minutes.
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Incuabate the denatured probe in 37°C water bath for 10-30 minutes for prehybridization.
- Wash the slide with 2X SSC for 5 minutes at room temperature.
- Pepsin treatment (optional):
- Incubate the slide in 0.01M HCl-diluted pepsin solution (10% w/v) at 37°C for 5 minutes.
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Wash the slide with 1X PBS for 5 min at room temperature twice.
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Wash the slide with 1X PBS/MgCl2 for another 5 minutes at room temperature.
- Dehydrate the slide with 70%, 85%, and 100% ethanol for 1 min each. Let the slide air dry.
- Denature the chromosome by incubating the slide in denaturing solution (70% formamide, 2X SSC, pH 7.0) for 5 minutes at 75°C.
- Dehydrate the slide with 70%, 85%, and 100% ethanol for 1 min each and air dry.
- Apply 10mL of denatured probe mix onto the slide.
- Immediately apply the coverslip and seal with rubber cement.
- Incubate the slide in a humidified chamber at 37°C for 3 days for hybridization.
Washing the slide and image visualization:
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Remove the rubber cement seal and the coverslip. Wash the slide with WSI (0.4X SSC, 0.3% Tween-20) at 75°C for 2 minutes.
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Wash the slide in WSII (2X SSC, 0.1% Tween-20) at room temperature for another 2 minutes.
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Dehydrate the slide with 70%, 85%, and 100% ethanol for 1 min each and air dry.
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Apply 40mL of DAPI II counterstain and place under dark until microscopic observation.
Data acquisition:
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The hybridized metaphase was observed under fluorescent microscopy. Image was captured under DAPI-, FITC- and Rhodamine-filters with the CCD camera. Usually, at least five images were captured randomly for one single case.
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The captured images were analysed and summarized using Vysis software. Eventually, a fluorescence intensity profile was obtained and the abnormal chromosomal regions were generally noticed.
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