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, Stefan K.G. Grebe

Research output: Contribution to journalArticle

30 Scopus citations

Abstract

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/μ1 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.

Original languageEnglish (US)
Pages (from-to)150-158
Number of pages9
JournalJournal of Molecular Diagnostics
Volume4
Issue number3
DOIs
StatePublished - Aug 2002

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Medicine

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