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JMD 2002, Vol. 4, No. 3
Copyright © 2002 American Society for Investigative Pathology & Association for Molecular Pathology

Loss of Heterozygosity Studies Revisited

Prior Quantification of the Amplifiable DNA Content of Archival Samples Improves Efficiency and Reliability

Kathryn Farrand^*, Lydija Jovanovic^*, Brett Delahunt^*, Bryan McIver, Ian D. Hay, Norman L. Eberhardt and Stefan K. G. Grebe^*

From the Department of Pathology and Molecular Medicine * Wellington School of Medicine and Health Sciences, Wellington, New Zealand; and the Departments of Medicine, and Molecular Biology and Biochemistry, Mayo Clinic and Foundation, Rochester, Minnesota

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/µl 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.

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