Step 1. Isolate DNA from FFPE samples
DNA from formalin-fixed/paraffin-embedded (FFPE) tissues is characterized by fragmentation and chemical modifications. Ambion's RecoverAll™ Total Nucleic Acid Isolation Kit helps overcome these limitations by maximizing the yield of DNA obtained from FFPE tissues.
Isolation of nucleic acids from archived tissue samples enables the retrospective study of diseased tissues at both the genomic and gene expression levels through the use of techniques like single nucleotide polymorphism (SNP) genotyping and Real-time PCR. While standard methods of tissue preservation are ideal for maintaining tissue structure and preventing putrefaction, they make it difficult to perform molecular analyses on samples. DNA from formalin-fixed/paraffin-embedded (FFPE) tissues is characterized by fragmentation and chemical modifications, such as protein-nucleic acid cross-links. This results in decreased template availability and can potentially compromise an SNP genotyping experiment.
The degree of fragmentation of DNA that has already occurred in FFPE tissues cannot be reversed. However, Ambion’s RecoverAll™ Total Nucleic Acid Isolation Kit yields sufficient DNA for successful SNP genotyping in many FFPE samples. FFPE tissue sections are first deparaffinized using a series of xylene and ethanol washes. The sections are then subjected to a rigorous protease digestion with an incubation time tailored for recovery of RNA or DNA. The nucleic acid is purified using a rapid glass filter methodology that includes an on-filter nuclease treatment. Finally, recovered DNA is eluted into either water or the low salt buffer provided.
>> Recommendation: Isolate human DNA from FFPE tissue using Ambion’s RecoverAll™ Total Nucleic Acid Isolation Kit.
In this study, researchers at Ambion, an Applied Biosystems business, addressed the feasability of obtaining accurate and reproducible SNP genotyping results using a wide variety of FFPE samples.
The RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE was used to isolate DNA from 109 human samples from a wide range of tissue types (colon, breast, uterus, kidney, tonsil, skin, muscle, pancreas, prostate, ovary, etc.) and block ages (1–18 years). A typical DNA isolation required less than 50 hours: 48 hours for protease digestion and 75 minutes hands-on processing time. Although DNA tends not to fragment as easily as RNA, it appears trapped to a greater degree by the formaldehyde tissue preservation process. Therefore, a longer (2 day) protease digestion treatment was required to release substantial amounts of DNA from these samples.