Validating Chl Ponchip results

A key step when using any type of microarray is the independent validation of the results. If RT-PCR is used to validate the results of an expression-microarray analysis, in the case of ChIP-on-chip experiments, individual single-ChIP assays should be performed to confirm the target sequences identified by this technique. It would be ideal to perform ChIPs with two different antibodies raised against the same protein. Specialized validating experiments are advisable. For instance, when we performed ChIP-on-chip analysis to investigate MBD targets in breast cancer cells (15), we validated the results by using both individual ChIP assays and a specific assay. In this case, since MBDs are known to associate specifically with methylated DNA (21), we investigated the methylation status of the CpG islands that each of the anti-MBD antibodies had been able to isolate. The specific methylation profile of each of the identified targets was an independent test that served not only to validate the results from the ChIP-on-chip analysis but also to reveal novel targets of epigenetic inacti-vation in human breast cancer. In the same system, for instance, when studying genes for which only one allele is methylated, an appropriate validating method is the coupling of individual ChIPs with bisulfite genomic sequencing (22). For nuclear factors that have a known or inferred binding site, it would be useful to search for that particular binding site in the positive clones resulting from the ChIP-on-chip experiment. Additionally, electrophoretic mobility-shift experiments can be used to test in vitro the ability to bind the resulting targets (23).

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