PCR amplification from paraffin-embedded tissues: recommendations on fixatives for long-term storage and prospective studies.
The development of polymerase chain reaction (PCR) DNA amplification methods has afforded molecular studies of fixed paraffin-embedded tissue samples and other archival material. Some fixation methods damage DNA, and thus deleteriously affect subsequent PCR analysis. This study addressed the effect of short- and long-term storage (2 hr to 30 days) in a variety of fixatives (10% buffered-neutral formalin [BNF], 95% ethanol, acetone, and OmniFix) before paraffin embedding. We tested the ability of prepared tissue sections to yield DNA amplification products ranging from 268 to 1327 bp. Results indicated that tissues fixed for 8 days in BNF were able to amplify 536-bp but very little 989-bp DNA fragments; after 30 days of BNF fixation only a 268-bp fragment was amplifiable. Samples fixed in OmniFix and acetone yielded products of 989 and 1327 bp, respectively, after 8 days of fixation; both fixatives yielded 989-bp amplification products after 30 days of fixation. Tissues fixed in 95% ethanol for up to 30 days efficiently produced DNA amplification fragments of up to 1327 bp in length. The results provide important information for prospective studies that involve PCR analysis from archival material. Furthermore, fixation and long-term storage in ethanol should prove particularly useful in remote areas where refrigeration or immediate sample-processing is unavailable.
Greer
C E
CE
Department of Infectious Diseases, Cetus Corporation, Emeryville, California 94608.
Lund
J K
JK
Manos
M M
MM
D004247:DNA / Q000235:genetics; D005069:Evaluation Studies as Topic; D005404:Fixatives; D006652:Histological Techniques; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D010232:Paraffin; D016133:Polymerase Chain Reaction / Q000379:methods*; D014021:Tissue Preservation
D005404:Fixatives; D010232:Paraffin; D004247:DNA