Obtaining material for hybridization

One important consideration in ChIP-on-chip experiments is the amount of immunoprecipitated DNA required for the hybridization. A standard ChIP DNA sample contains a variable amount of DNA of between 50 and several hundred nanograms. In a ChIP-on-chip experiment, between 1 and 2 ^g of immunoprecipitated material is required for a single hybridization experiment. Two approaches have been taken to overcome this limitation and obtain the required amount. Firstly, it is possible to scale up the ChIP experiment, which generally means increasing the amounts of cells and of antibody. However, in some cases, both these quantities are limited, and an alternative approach is possible that exploits random PCR amplification of the immunoprecipitated material.

In the first case, it is best to perform multiple single-standard ChIP assays rather than amplifying the volume in a single experiment. Usually, 30 single IP experiments should yield enough material for hybridization. Samples should be treated and processed separately, and only after DNA samples have been resuspended in water should they be combined to proceed with fluorescent labeling and hybridization.

When the amount of biological material or the availability of the antibody is limited, it is possible to use an amplification step. This protocol has been described by Kuukasjarvi et al. (19) and modified by Huang et al. (20). Basically, two consecutive amplification steps are performed. The first requires the use of Thermosequenase, a degenerate primer (5'-CCG ACT CGA GNN NNN NAT GTG G-3') and low-stringency amplification conditions (3 min at 94°C, followed by four cycles of 1 min at 94°C, 1 min at 25°C, 3 min transition at 25-74°C, 2 min extension at 74°C, and a final extension of 10 min). The second step consists of a more standard PCR amplification, standard Taq polymerase and more stringent conditions are used (3 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 56°C, 2 min extension at 72°C, and a final extension of 10 min). It is important to run a confirmatory gel. There should be a smear of DNA, of length between 300 and 1000 bp, present for the antibody-treated samples. Negative controls should be added for each DOP-PCR step in order to rule out the existence of non-specific amplification of contaminant DNA.

Before labeling and hybridizing the ChIP samples, it is advisable to test a small aliquot of the samples for PCR amplification of a positive and negative control for both antibody-treated and no-antibody samples. This test depends on the availability of known in vivo binding targets for the protein of interest.

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