The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when?
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.
Gilbert
M Thomas P
MT
Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona, United States of America. mtpgilbert@gmail.com
Haselkorn
Tamara
T
Bunce
Michael
M
Sanchez
Juan J
JJ
Lucas
Sebastian B
SB
Jewell
Laurence D
LD
Van Marck
Eric
E
Worobey
Michael
M
D000293:Adolescent; D000328:Adult; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D005404:Fixatives / Q000737:chemistry*; D006358:Hot Temperature; D006801:Humans; D016651:Lithium Carbonate / Q000494:pharmacology; D008168:Lung / Q000378:metabolism; D008223:Lymphoma / Q000235:genetics; D008875:Middle Aged; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification*; D012514:Sarcoma, Kaposi / Q000235:genetics; D013154:Spleen / Q000378:metabolism; D016707:Tissue Fixation; D055815:Young Adult
D005404:Fixatives; D016651:Lithium Carbonate; D012313:RNA; D004247:DNA