Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications.
BACKGROUND
A large portion of tissues stored worldwide for diagnostic purposes is formalin-fixed and paraffin-embedded (FFPE). These FFPE-archived tissues are an extremely valuable source for retrospective (genetic) studies. These include mutation screening in cancer-critical genes as well as pathogen detection. In this study we evaluated the impact of several widely used DNA extraction methods on the quality of molecular diagnostics on FFPE tissues.


RESULTS
We compared 4 DNA extraction methods from 4 identically processed FFPE mammary-, prostate-, colon- and lung tissues with regard to PCR inhibition, real time SNP detection and amplifiable fragment size. The extraction methods, with and without proteinase K pre-treatment, tested were: 1) heat-treatment, 2) QIAamp DNA-blood-mini-kit, 3) EasyMAG NucliSens and 4) Gentra Capture-Column-kit.Amplifiable DNA fragment size was assessed by multiplexed 200-400-600 bp PCR and appeared highly influenced by the extraction method used. Proteinase K pre-treatment was a prerequisite for proper purification of DNA from FFPE. Extractions with QIAamp, EasyMAG and heat-treatment were found suitable for amplification of fragments up to 400 bp from all tissues, 600 bp amplification was marginally successful (best was QIAamp). QIAamp and EasyMAG extracts were found suitable for downstream real time SNP detection. Gentra extraction was unsuitable. Hands-on time was lowest for heat-treatment, followed by EasyMAG.


CONCLUSIONS
We conclude that the extraction method plays an important role with regard to performance in downstream molecular applications.
Huijsmans
Cornelis Jj
CJ
Laboratory of Molecular Diagnostics, Jeroen Bosch Hospital, 's-Hertogenbosch, The Netherlands. r.huysmans@jbz.nl.
Damen
Jan
J
van der Linden
Johannes C
JC
Savelkoul
Paul Hm
PH
Hermans
Mirjam Ha
MH