The Isolation of Nucleic Acids from Fixed, Paraffin-Embedded Tissues–Which Methods Are Useful When?

TLDR

Museums and pathology collections worldwide hold archived genetic material preserved in formalin, which cross‑links biomolecules and hampers nucleic acid isolation and PCR. The study aims to quantitatively compare nucleic acid quality from fixed specimens across multiple published protocols. The authors tested various pre‑treatment, extraction, purification, and post‑extraction enzymatic repair methods on a panel of fixed tissues. Results show that while some methods improve certain nucleic acid attributes, many offer no benefit and can incur trade‑offs, so protocol choice must be tailored to specific goals, with a reference table provided.

Abstract

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.