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As discussed above all the steps in a CGH experiment are critical to a successful outcome. Furthermore, many of them need to be adjusted until satisfactory results can be reliably obtained. In the following discussion I will highlight the particular problem areas and the experimental manoeuvres used to try to prevent them.

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2.2.1.9.1 Preparation o f Metaphase spreads

The quality of the slides is probably the single most important factor in determining the success of a CGH experiment (Karhu, R. et a l 1997), and there are a number of variables which need to have particular attention paid to them. The chromosome size and quantity of metaphases is variable. Colcemid times longer than 20min will give more metaphase spreads but the chromosomes will be more condensed. Therefore a balance needs to be achieved between chromosome length and number. One way of obtaining numerous chromosomes of a certain size is to synchronise the cell culture, for example with methotrexate, which blocks mitosis. However, this is something which we never found to be necessary; optimising the colcemid time at 15 minutes was sufficient.

When fixing the cells, it is most important to add the first fix slowly. If it is added too quickly, it will result in the pellet clumping, and poor fixation. Proper fixation ensures crisp, well-spread chromosomes. Placing the cells at 4°C also helps facilitate fixation, and so during preparation the fixed material was left in second fix overnight. The more times the pellets are washed with fixative the cleaner the final preparation will be, with less cytoplasm, so all my pellets were washed between 5 and 10 times.

Spreading of the metaphase chromosomes is variable. Ideally metaphases should be well spread with not too many overlapping chromosomes. Metaphases spread well when they are well fixed, as discussed above and when the microscope slides are clean, hence the importance of storing them in ethanol with a small amount of hydrochloric acid and drying them, just prior to dropping the spreads, with lint free tissue. The other important factor is the atmospheric conditions. However, conditions can vary considerably (temperature and humidity) and so if the metaphase spreads did not look optimal on one day, the fixed material was stored at 4°C and an attempt made to prepare them on another day. The conditions described above are the ones which we found to work well in our laboratory. If the conditions are too cold/dry the metaphases will not spread.

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Conversely, if the conditions are too hot/humid the metaphases overspread and indeed in extreme cases appear as a ‘chromosome soup’.

The amount of cytoplasm is critical. If the metaphases appear to be embedded in too much cytoplasm then washing the pellet and/or the slides with more fixative can be tried. The amount of cytoplasm is critical as too much prevents both optimal dénaturation and probe penetration, with a resulting poor hybridisation. However a small amount of cytoplasm may allow the chromosomes to withstand the necessary dénaturation. Some authors pretreat the slides with proteinase K or pepsin (Courjal, F. and Theillet C. 1997) to remove excess cytoplasm, but I found that if the slides are of sufficient quality pretreatment is not necessary. Indeed pretreatment with pepsin using the conditions recommended by other authors resulted in overdenaturation with loss of the chromosome structure. We therefore abandoned this strategy. If a batch o f slides has a large amount of cytoplasm, they will not be useful for CGH and so should be discarded for CGH use. However, they are usually of sufficient quality for use in FISH experiments. Although it is time consuming to prepare a new batch, it is ultimately worthwhile compared to setting up many CGH experiments which yield doubtful results. If a new batch has to be prepared a longer hypotonic stage may be helpful, in reducing the amount of cytoplasm.

It is necessary to test the quality of each batch of slides, prior to use; again if they are of insufficient quality then they should be discarded. Slides were quality tested by denaturing them (using a range o f dénaturation times e.g. 2-5 nun), staining with DAPI and then examining them under the microscope. This is necessary as slides which look satisfactory down the microscope may not necessarily withstand dénaturation well. Optimal metaphase spreads are ones which tolerate enough dénaturation to achieve uniform hybridisation along each chromosome but do not lose their structure and morphology. By implication from this, slides which are underdenatured will not hybridise and if overdenatured the chromosomes appear fat without normal morphology. Once an optimal dénaturation time has been defined the slides should have a test hybridisation with normal male and female DNA; all autosomes should have a ratio between 0.9 and 1.1 and the X chromosome should have a ratio of <0.6 see

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Figure 2-4 and Figure 2-5. Although clearly the sex chromosome ratio should be less than 0.5, this is something which is rarely seen (Karhu, R. et al. 1997).

Within each experiment a control was set up with normal male (green) and normal female DNA (red). The fluorescence ratios, particularly o f the X chromosome should be as discussed above. If using DOP-PCR to label DNA a useful internal control for an experiment is to pair tumour DNA with DNA from the opposite sex. The profile of the X chromosome will be a guide to the quality of the experiment, i.e. a ratio which is less than expected will suggest a poor quality experiment.

2.2.1.9.2 Probe preparation

The size and quality of the probe is an important step in determining the quality of the CGH. If the probe fragments are too large then the incubation time should be increased or more enzyme (DNase/Polymerase mix) added, if the fragment size is too small then the experiment should be repeated with a shorter incubation time or by the addition of less enzyme. If the probes are too large then the resulting hybridisation will be poor with typically a spotty or speckled appearance. DNA contaminated with protein will label inefficiently and give a similar hybridisation appearance. A large amount of contaminating protein will mean that the probe will fail to nick at all and in this situation the DNA needs to be repurified.

Some authors use less than Ipg of nick translated labelled probe, however in our experience this amount gives a strong uniform hybridisation. However if using DOP-PCR labelled products, using less probe may be unavoidable.

2 .2 .1.9.3 DOP-PCR

DOP-PCR is a difficult technique. It is often not very reproducible and is prone to contamination. Separate pipettes should be used for PCR only. If possible it should be done in a dedicated room or area with surfaces, pipettes and tubes

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subjected to UV irradiation. If contamination occurs, this should be dealt with by preparing fresh stock solutions and cleaning the pipettes used and taking the usual precautionary methods for any PCR. Although such DNA run on a gel will look satisfactory, it is only when it is hybridised that the problem will become manifest. If the DOP-PCR still fails to work different magnesium concentrations can be tried, some authors use PCR buffer containing 20mM magnesium. A recent paper recommending an alternate protocol for DOP-PCR (Kuukasjarvi, T.

et al. 1997c), was not found to be helpful, as it gave very large fragment smears,

which did not hybridise well (see Appendix 1). The unique problems which arise when the tissue has been embedded in a fixing medium will be discussed separately in Appendix 1. An alternative approach for DOP-PCR is to use a kit which is now commercially available (Boehringer cat no: 1644 963).

2.2.1.9.4 C otl and Preannealing times

The amount of Cot I and preannealing times are important. If there is insufficient suppression of the repetitive sequences this will result in strong staining of the heterochromatin regions of chromosomes 1,9, 16 and 19 and high intensity of non-specific fluorescence at chromosomal regions which have a high content of repetitive sequences. Both these changes cannot be corrected for during image analysis and will result in making it difficult to detect small copy number alterations in the heterochromatin regions and inaccuracies in the fluorescence ratio in regions of repetitive sequences.

2.2.1.9.5 Post-hybridisation washes

The post hybridisation washes are probably subject to the most variability between different authors. However, those outlined here give good results with a very good signal to noise ratio. Use of more stringent washes for example 0.1% SSC resulted in considerable loss of signal.