DNA amplification by polymerase chain reaction from brain tissues embedded in paraffin.
A method which enables analysis of DNA from archival paraffin embedded normal and malignant brain tissue is described. The demonstration of a 317-bp long beta-actin DNA sequence by the polymerase chain reaction (PCR) was used to identify which fixation procedure, deparaffinization time and DNA extraction procedure would give the best results. Tissue specimens 1-39 years old were included in the experiments. Specimens fixed in either 10% formalin, Carnoy's or AMeX fixative were found to be best suited for subsequent analysis by PCR. Paraformaldehyde and acetone compromised amplification efficiency, while Bouin's fixed tissue gave uniformly negative results. Regardless of fixative used, PCR reaction had to be run through at least 40 cycles. Prolonged deparaffinization time and phenol/chloroform extraction of DNA did not influence DNA quality as a template for PCR reaction. Formalin fixed brain tumours can be successfully used for DNA/PCR analysis even if they are up to 39 years old.
Department of Molecular Medicine, Ruder Bosković Institute, Zagreb, Croatia.
K
K
Gall
J
J
Pavelić
D
D
Jadro-Santel
M
M
Poljak
K
K
Pavelić
D001483:Base Sequence; D001923:Brain Chemistry; D001932:Brain Neoplasms / Q000235:genetics*; D004247:DNA / Q000032:analysis*; D005404:Fixatives / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D013997:Time Factors; D016707:Tissue Fixation / Q000379:methods
D005404:Fixatives; D004247:DNA