Optimising restriction enzyme cleavage of DNA derived from archival histopathological samples: an improved HUMARA assay.
OBJECTIVE
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.
Jovanovic
Lidija
L
Department of Pathology and Molecular Medicine, Wellington School of Medicine and Health Sciences, Wellington, New Zealand.
Delahunt
Brett
B
McIver
Bryan
B
Eberhardt
Norman L
NL
Grebe
Stefan K G
SK
D003106:Colon / Q000737:chemistry; D004247:DNA / Q000032:analysis / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005260:Female; D005557:Formaldehyde; D006801:Humans; D008297:Male; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D011944:Receptors, Androgen / Q000032:analysis / Q000378:metabolism*; D015183:Restriction Mapping / Q000379:methods*; D013961:Thyroid Gland / Q000737:chemistry; D016707:Tissue Fixation
C410798:AR protein, human; D011944:Receptors, Androgen; D005557:Formaldehyde; D004247:DNA; C024406:endodeoxyribonuclease HpaI; D015252:Deoxyribonucleases, Type II Site-Specific; C037130:GCGC-specific type II deoxyribonucleases