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Our aim was to evaluate and optimise methylation-sensitive restriction enzyme assays for use in formalin-fixed, paraffin-embedded tissue (FFPET) samples, in order to improve the application of the HUMARA X-chromosome inactivation assay to FFPET samples.We extracted DNA from normal male colon and thyroid FFPET. Several DNA clean-up procedures and restriction enzyme buffer compositions were tested for two methylation-sensitive enzymes, HhaI and HpaII and a non-methylation-sensitive isoschizomere, MspI.By including both a non-methylation-sensitive control enzyme and DNA from male archival specimens in our experiments, we were able to detect even subtle degrees of incomplete digestion. We showed that FFPET-derived DNA is a poor substrate for restriction enzymes, especially for methylation-sensitive restriction endonucleases. An optimised DNA clean-up protocol and restriction enzyme buffer-mix allowed us to achieve complete digestion.The combination of multiple, rigorous controls, DNA clean-up and restriction buffer optimisation increases the reliability of HUMARA-based X-chromosome inactivation analysis of FFPET samples. Analogous approaches are likely to allow optimisation of other restriction enzyme-based assays of FFPET samples.

Polymerase chain reaction (PCR)-based loss of heterozygosity (LOH) studies of archival formalin-fixed, paraffin-embedded (FFPE) tumor tissues have become an important tool in the search for tumor suppressor genes and oncogenes and are also used increasingly in clinical practice. However, FFPE tissue samples may contain little amplifiable DNA, resulting in frequent reaction failures and unreliable LOH data. Using pairs of serial dilutions of reference DNA, we determined the minimum amplifiable DNA concentration necessary for reliable microsatellite-PCR LOH analysis. We then measured the amplifiable DNA content of a selection of frozen and FFPE-derived tumor specimens by real-time quantitative PCR. A minimum input of 600 pg of 100% amplifiable DNA per PCR was required for reliable LOH analysis. While the total DNA concentrations of all samples exceeded this figure, most FFPE-sample-derived DNA was non-amplifiable, with ratios of actually amplifiable DNA to total DNA as low as 1 to 3625. Many FFPE samples therefore contained substantially less than 600 pg/microl of actually amplifiable DNA, making them potentially unsuitable for LOH studies. Real-time quantitative PCR before LOH studies of FFPE tissues allows: identification of samples, which will fail microsatellite-PCR; exclusion of samples, which will yield unreliable results; and optimal adjustment of template input for the remainder. Amplification reactions undertaken without this precaution can result in unreliable LOH data.