The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when?
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The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when? / Gilbert, M Thomas P; Haselkorn, Tamara; Bunce, Michael; Sanchez, Juan J; Lucas, Sebastian B; Jewell, Laurence D; Van Marck, Eric; Worobey, Michael.
In: PLoS ONE, Vol. 2, No. 6, 2007, p. e537.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when?
AU - Gilbert, M Thomas P
AU - Haselkorn, Tamara
AU - Bunce, Michael
AU - Sanchez, Juan J
AU - Lucas, Sebastian B
AU - Jewell, Laurence D
AU - Van Marck, Eric
AU - Worobey, Michael
PY - 2007
Y1 - 2007
N2 - 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.
AB - 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.
U2 - 10.1371/journal.pone.0000537
DO - 10.1371/journal.pone.0000537
M3 - Journal article
C2 - 17579711
VL - 2
SP - e537
JO - PLoS ONE
JF - PLoS ONE
SN - 1932-6203
IS - 6
ER -
ID: 3848462