FFPE (formalin-fixation paraffin-embedding) is an important method of sample preservation and preparation – particularly in the context of cancer research and next-generation sequencing (NGS). It’s essential for preserving biopsy specimens that would otherwise decay in vitro or in storage.

When a biopsy is formalin-fixed, it’s treated with formaldehyde which preserves the key tissue structures. It’s then embedded into a waxy paraffin block and sectioned using a microtome. This fixation and embedding method has proven invaluable for therapeutic purposes for decades, and it enables scientists to archive samples from different patients to facilitate future research. However, challenges arise when it comes to isolating DNA from FFPE tissues.

DNA Isolation from FFPE Tissues: Challenges

Regardless of the eventual area of application, all FFPE processes begin with tissue extraction either from patients, or animals in the lab. Biopsied tissue is excised and immersed in a formalin buffer for up to 24 hours. This is then dehydrated before the specimen is embedded in paraffin within a mould, forming an FFPE block. This prevents the decay of the biopsied tissue, preserving it for storage and future analysis. In a clinical setting, pathologists are then able to section these blocks and inspect the tissue under the microscope to determine if abnormalities are present. In a research setting, where the goal might be to characterise the underlying genetic abnormalities of a particular cancer, the first step generally involves DNA or RNA isolation.

One of the chief issues of DNA and RNA extraction from FFPE tissue is the prevalence of chemical crosslinking during formalin-fixation. This cross-linking binds nucleic acids to other proteins and other DNA strands, complicating the isolation process. Ultimately, if steps aren’t taken to decouple these cross links, isolation will yield impure, degraded and fragmented DNA. This has repercussions for downstream analysis such as PCR, qPCR and NGS, as they require high quality starting material to produce accurate results.

On top of crosslink decoupling, FFPE tissues must be properly deparaffinised so paraffin does not contaminate the sample. Typically, harsh solvents such as xylene are used in this process which exposes personnel to hazardous materials.

Both qPCR and NGS applications demand a solution to the challenges represented by FFPE tissues.

Solutions for DNA Isolation from FFPE Tissues

Mediray offers a selection of tailored DNA isolation kits specifically attuned to the challenges of FFPE tissues. Our product range forgoes the use of xylene in deparaffinization, relying instead on a proprietary paraffin dissolver for simple and convenient removal of supportive paraffin from FFPE slices or tubes. This patented solution is compatible for both DNA and RNA extraction processes and is a mild solution that doesn’t require the use of fume extractors to ensure safe operating conditions.

Macherey-Nagel’s selection of  NucleoMag and Nucleospin FFPE extraction kits operate on either silica-membrane or magnetic-bead technologies, employing the patented Paraffin Dissolver, allowing efficient lysis in a convenient two-phase system. These proprietary buffers assist in the shearing of DNA into consistent fragment sizes, overcoming formalin cross-linking over a short incubation period. This guarantees the highest quality and highest yield of DNA or RNA possible for given FFPE samples.

Macherey-Nagel’s DNA isolation FFPE kits are applicable for downstream PCR and NGS, supporting ongoing innovations in various biochemistry, life sciences, and therapeutic applications. If you would like to learn more about what this means for your application, please don’t hesitate to contact us today.

Journal References:

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098187

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3970103/

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197456

Additional References:

https://www.nature.com/articles/6602889

https://www.promega.co.uk/resources/pubhub/overcoming-challenges-of-dna-purification-from-ffpe-samples/