The effect of fixation type on DNA extracted from paraffin-embedded tissue for PCR studies in dermatopathology.
BACKGROUND
Amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction (PCR) is widely used to detect viral genomes, clonal gene rearrangements and oncogene mutations in skin specimens. Fixation with embedding of skin tissue is a procedure that has a profound effect on its molecular arrangement.


OBJECTIVE
The aim of this study was to determine the effect of different fixatives on the PCR amplification of DNA.


METHODS
We fixed randomly chosen fresh pathologic skin specimens in formalin, ethanol and Histochoice for 24 and 72 h and then embedded the tissue in paraffin. DNA was extracted from the paraffin-embedded tissues and used as template for amplification, producing 530- and 760-bp fragments of the phosphoglycerokinase gene.


RESULTS
Our results indicate that PCR can be performed with excellent results on ethanol- and Histochoice-fixed, paraffin-embedded skin tissue with a rate of success comparable to that using fresh tissues; formalin-fixed tissue gave slightly less satisfactory results.


CONCLUSIONS
This investigation corroborates previous reports investigating the effect of ethanol and formalin fixation on DNA amplification by PCR. Moreover, this is the first study showing that DNA extracted from tissue fixed with Histochoice is suitable for PCR gene amplification.
Alaibac
M
M
Laboratory of Experimental Oncology, National Cancer Institute, Bari, Italy.
Filotico
R
R
Giannella
C
C
Paradiso
A
A
Labriola
A
A
Marzullo
F
F
D001483:Base Sequence; D003470:Culture Media; D046508:Culture Techniques; D004247:DNA / Q000187:drug effects* / Q000302:isolation & purification*; D003880:Dermatology / Q000379:methods; D000431:Ethanol / Q000494:pharmacology; D005404:Fixatives / Q000494:pharmacology*; D005557:Formaldehyde / Q000494:pharmacology; D005784:Gene Amplification; D006801:Humans; D008969:Molecular Sequence Data; D009696:Nucleic Acids / Q000378:metabolism; D010232:Paraffin; D010592:Pharmaceutic Aids / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000187:drug effects*; D011897:Random Allocation; D012016:Reference Values; D012867:Skin / Q000473:pathology*; D012997:Solvents / Q000494:pharmacology
D003470:Culture Media; D005404:Fixatives; D009696:Nucleic Acids; D010592:Pharmaceutic Aids; D012997:Solvents; D005557:Formaldehyde; D000431:Ethanol; D010232:Paraffin; D004247:DNA