Optimising restriction enzyme cleavage of DNA derived from archival histopathological samples: An improved HUMARA assay

Lidija Jovanovic, Brett Delahunt, Bryan McIver, Norman L. Eberhardt, Stefan K.G. Grebe

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

Aims: 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. Methods: 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. Results: 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. Conclusions: 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.

Original languageEnglish (US)
Pages (from-to)70-74
Number of pages5
JournalPathology
Volume35
Issue number1
DOIs
StatePublished - Feb 2003

Keywords

  • Archival histopathological samples
  • Assay optimisation
  • DNA purification/clean up
  • Enzyme inhibitors
  • Formalin-fixed paraffin-embedded tissues
  • HUMARA assay
  • Restriction enzyme assays
  • Tumor clonality
  • Tumour clonality
  • X-chromosome inactivation

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

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