Feasibility of archival non-buffered formalin-fixed and paraffin-embedded tissues for PCR amplification: an analysis of resected gastric carcinoma.
Although several factors affecting the sensitivity of polymerase chain reaction (PCR) amplification from formalin-fixed tissues have been investigated mostly by experiments, the feasibility of archival formalin-fixed, paraffin-embedded tissue samples stored in pathology departments for PCR amplification has rarely been examined directly. Thus, the feasibility of 74 archival unbuffered 10% formalin-fixed, paraffin-embedded tissues for PCR amplification with primers producing a 190 b.p. DNA segment of p53 exon 5 was investigated. Fixation time was the critical factor influencing the sensitivity of PCR amplification. All (6/6) of the samples fixed for only 1 day, 44% (7/16) of the samples fixed for 2-3 days and 14% (4/28) of the samples fixed for 4-6 days showed successful amplification, while no amplification was obtained for the samples fixed for 7 days or more. The peak size of DNA extracted from the archival tissues decreased as the fixation time became longer. Experiments using xenografted tumor tissues fixed for various times showed longer permissible fixation time; up to 9 days of fixation, decreasing amounts of PCR products were obtained while no amplification was obtained for the samples fixed for 12 days or more. The time in paraffin seemed to be a minor factor for PCR amplification since all of the 1 day fixation samples, including those that had been embedded for up to 5 years, resulted in efficient amplification. The size of the amplified DNA segments, however, could be another factor influencing the sensitivity of amplification because even the 1 day fixation samples showed less amplification of 345 b.p. DNA compared with those of 167 and 262 b.p. DNA. Additionally, a point mutation was detected in the amplified p53 products from archival tissues using a non-isotopic method, temperature gradient gel electrophoresis. In conclusion, archival tissue samples that had been fixed immediately for only up to 1 day were constantly available for PCR amplification of approximately 200 b.p. DNA segments, suggesting that surgical specimens should be subjected to cutting and paraffin embedding just after 1 day or less fixation for subsequent use in PCR amplification.
Inoue
T
T
Department of Pathology, Miyazaki Medical College, Japan.
Nabeshima
K
K
Kataoka
H
H
Koono
M
M
D000818:Animals; D002277:Carcinoma / Q000235:genetics* / Q000473:pathology / Q000601:surgery; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000032:analysis* / Q000187:drug effects; D004587:Electrophoresis, Agar Gel; D005557:Formaldehyde; D005561:Formates / Q000494:pharmacology; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods*; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D013274:Stomach Neoplasms / Q000235:genetics* / Q000473:pathology / Q000601:surgery; D013997:Time Factors; D016707:Tissue Fixation; D014183:Transplantation, Heterologous
D017931:DNA Primers; D004273:DNA, Neoplasm; D005561:Formates; C030544:formic acid; D005557:Formaldehyde