The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when?
The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when?
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DOI：
10.1371/journal.pone.0000537
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发表时间：
2007-06-20
期刊：
影响因子：
3.7
通讯作者：
Worobey M
中科院分区：
文献类型：
--
作者：
Gilbert MT;Haselkorn T;Bunce M;Sanchez JJ;Lucas SB;Jewell LD;Van Marck E;Worobey M
世界各地的博物馆和病理学收藏品是研究人口和疾病的遗传材料档案。出于保存的目的，这些收藏品中的很大一部分被固定在含有福尔马林的溶液中，这种处理导致生物分子的交联。交联不仅使核酸的分离复杂化，而且在PCR期间引入聚合酶“阻断”。从档案组织中回收DNA和RNA的方法多种多样，尽管以前的一些研究定性地比较了不同技术的相对优点，但很少进行大规模的定量比较。为了帮助解决这一问题，我们进行了一项研究，调查从固定标本的测试面板中回收的核酸的质量，这些标本已按照许多已发表的方案进行操作。这些包括在提取之前预处理样品的方法、提取和核酸纯化方法本身以及提取后酶修复技术。我们发现，尽管许多已发表的方法对核酸的某些特征具有明显的积极影响，但这些好处往往是有代价的。此外，许多以前发表的技术似乎根本没有效果。我们的研究结果建议，应谨慎选择所采用的提取方法。在这里，我们提供了一个快速参考表，可用于确定特定目标的适当协议。
Museums and pathology collections around the world represent an archive of genetic material to study populations and diseases. For preservation purposes, a large portion of these collections has been fixed in formalin-containing solutions, a treatment that results in cross-linking of biomolecules. Cross-linking not only complicates isolation of nucleic acid but also introduces polymerase “blocks” during PCR. A wide variety of methods exists for the recovery of DNA and RNA from archival tissues, and although a number of previous studies have qualitatively compared the relative merits of the different techniques, very few have undertaken wide scale quantitative comparisons. To help address this issue, we have undertaken a study that investigates the quality of nucleic acids recovered from a test panel of fixed specimens that have been manipulated following a number of the published protocols. These include methods of pre-treating the samples prior to extraction, extraction and nucleic acid purification methods themselves, and a post-extraction enzymatic repair technique. We find that although many of the published methods have distinct positive effects on some characteristics of the nucleic acids, the benefits often come at a cost. In addition, a number of the previously published techniques appear to have no effect at all. Our findings recommend that the extraction methodology adopted should be chosen carefully. Here we provide a quick reference table that can be used to determine appropriate protocols for particular aims.
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影响因子：
2.9
作者：
BRUTLAG, D;SCHLEHUB.C;BONNER, J
通讯作者：
BONNER, J
影响因子：
3.1
作者：
Benavides, Julio;Garcia-Pariente, Carlos;Perez, Valentin
通讯作者：
Perez, Valentin
影响因子：
--
作者：
Longy, M;Duboue, B;Moynet, D
通讯作者：
Moynet, D
影响因子：
3.1
作者：
BONI, J;SCHUPBACH, J
通讯作者：
SCHUPBACH, J
影响因子：
3.4
作者：
Coura, R;Prolla, JC;Ashton-Prolla, P
通讯作者：
Ashton-Prolla, P