Technique / Molecular Biology / PCR / Standard PCR

Paraffin embedded tissue PCR


Paraffin embedded tissue PCR

PCR Methods and Applications, Vol 1, 46-50
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ARTICLES


PCR amplification from paraffin-embedded tissues: recommendations on fixatives for long-term storage and prospective studies
CE Greer, JK Lund and MM Manos
Department of Infectious Diseases, Cetus Corporation, Emeryville, California 94608.

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.

Last update 23-Aug-2002, Rating Good of 1 votes.

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By T&M on 21-Nov-2005
for pretreatment of tissues you can give following treatment:
1. 15-20 hour incubation with Xylene
2. washing with absolute alcohol
3.incubation at 65 degrees for 15 min
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By Merijn on 27-Sep-2004
Dneasy works fine with me but I add 10 ul of fresh 15% DTT (dithiotreitol) to the extraction mix. It is supposed to breakdoen the sulfer bounds in the creatine of the feather. I use only the camalus (the tip of the feather which is normaly in the birds body) of the feathers.
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By biologa on 23-Aug-2002
hi, I work sexing birds, by pcr and electroforesis, and I have benn having problems extracting dna from feathers, is anybody familiar with this problem? and I was thinking about using adneasy extraccion kit, to extract dna, can anybody give some advise?
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