The suitability of DNA extracted from formalin-fixed, paraffin-embedded tissues for double differential polymerase chain reaction analysis.
Formalin-fixed, paraffin-embedded (FFPE) tissues are one of the popular sources of diagnostic materials, the easiest to store and transport. They are often used as the source of nucleic acids for retrospective molecular analyses based on DNA amplification by polymerase chain reaction (PCR). However, it is known that nucleic acids from paraffin-embedded tissues are much worse templates than those recovered from fresh tissues. It is exceptionally important in a quantitative analysis, including double differential PCR (ddPCR). Therefore, a pilot study comparing quantity and quality of DNA extracted with various paraffin removal and DNA isolation procedures from FFPE tissues was conducted. Furthermore, the suitability of DNA isolated with optimized procedure for the assessment of erbB-2 average gene copy number (AGCN) was checked. Specimens for comparison of extraction and isolation procedures were generated from the same human normal thyroid tissue embedded in paraffin to eliminate variabilities in tissue processing and sample size. Three procedures of paraffin removal and three procedures of DNA extraction from deparaffinized tissue were compared. Only one procedure provided DNA, which was efficiently amplified in ddPCR. The material obtained with this optimized procedure was used to check the precision of ddPCR by evaluation of AGCN of erbB-2 oncogene. Low variability of obtained results close to expected AGCN value (AGCN=1) indicates high reproducibility of the method, as well as its high accuracy, if the normal value of erbB-2 AGCN in the examined tissue is assumed.
Bielawski
K
K
Molecular Diagnostics Division, Department of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland. krzysztof_bielawski@wp.pl
Zaczek
A
A
Molecular Diagnostics Division, Department of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland. krzysztof_bielawski@wp.pl|Postgraduate School of Molecular Medicine, Warsaw, Poland
Lisowska
U
U
Molecular Diagnostics Division, Department of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland. krzysztof_bielawski@wp.pl
Dybikowska
A
A
Molecular Diagnostics Division, Department of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland. krzysztof_bielawski@wp.pl
Kowalska
A
A
Molecular Diagnostics Division, Department of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland. krzysztof_bielawski@wp.pl
Falkiewicz
B
B
Molecular Diagnostics Division, Department of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland. krzysztof_bielawski@wp.pl
D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D005404:Fixatives; D005557:Formaldehyde; D018628:Gene Dosage; D005914:Globins / Q000235:genetics; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013482:Superoxide Dismutase / Q000235:genetics; D016707:Tissue Fixation / Q000379:methods
D004247:DNA; D005404:Fixatives; D005557:Formaldehyde; D005914:Globins; D018719:Erb-b2 Receptor Tyrosine Kinases; D013482:Superoxide Dismutase