Formalin-fixed, paraffin-embedded (FFPE) samples are invaluable resources for molecular pathology and biomedical research, preserving tissue architecture and enabling long-term storage. However, the very fixation process that protects these samples often complicates the extraction of high-quality nucleic acids, especially when over-fixation occurs. FFPE tissues present unique challenges, such as cross-linking, nucleic acid fragmentation, and the limited amount of precious sample available, that can compromise downstream applications such as PCR, sequencing, and gene expression analysis. In this post, we’ll explore the common hurdles scientists face when working with FFPE samples, including a first-hand account from one of our customers, and share options for optimizing your extraction workflow. Whether you’re a seasoned scientist or new to working with FFPE tissues, this post will equip you with the knowledge to enhance your experimental success.
1. Understanding the Limits of FFPE Samples
Formalin-Fixed Paraffin-Embedded (FFPE) samples are invaluable resources in molecular biology and pathology, preserving tissue morphology and enabling long-term storage. However, the process of fixation can introduce significant challenges when it comes to extracting high-quality nucleic acids. Over-fixation occurs when tissue samples are exposed to formalin for extended periods, leading to excessive cross-linking of proteins and nucleic acids. This results in fragmentation and chemical modification of DNA and RNA, making them more difficult to recover and compromising their integrity. Consequently, over-fixed FFPE samples often yield lower quantities of nucleic acids, if they yield any at all, and those that are recovered are typically more degraded and less suitable for downstream applications. Understanding these impacts is crucial, as it informs the selection of extraction protocols and modifications needed to maximize the quality and utility of nucleic acids obtained from these challenging samples.
2. A Researcher’s Experience with Automating FFPE Extractions
Resident Laboratory Ltd is a molecular pathology lab based out of Romania that specializes in diagnostics and paternity testing. Dr. Ferenc Fazakas, a geneticist and co-founder of Resident Laboratory Ltd., is no stranger to the challenges of working with FFPE specimens in his pathology lab. “Doing assays on FFPE samples, [we would] probably face a failure rate [between] 1% and 5%. It can be hard to accept that a sample of a big chunk of tissue can still give you nothing in the tube,” he says.
Still, Dr. Fazakas didn’t give up hope that his FFPE extractions could be optimized with a new layout and automation, “When our sample numbers were kind of small or reduced, single tube or semi-automatic extractions were mainly used, but after a while, as the sample number grew, we had to change to a different method. We changed to microplates instead of strips or single tubes which have several advantages like reducing the sample change. In our lab, there is only one point where the samples can be placed in the wrong place; otherwise, we are using automation, and the placement of the samples is maintained throughout the whole process. Moreover, we started doing RNA assays as well and needed to do RNA extraction from the same samples as DNA extraction. So, Omega [Bio-tek] was quite flexible from this point of view because their kit allows sequential extraction.”
Dr. Fazakas’ experience highlights that while overly-fixed FFPE samples present formidable challenges, it is still possible to achieve an efficient DNA and RNA extraction workflow that unlocks the valuable molecular data stored in these specimens. He continues, “If the sample is wrong or over-fixed, the result will be wrong, too, but Omega [Bio-tek] provided flexibility and a really easy way of automating [our workflow]. Moreover, the RNA as a second extraction step made it obvious to choose Omega [Bio-tek].”
3. Techniques for Improved Extraction Workflows
Extracting high-quality DNA and RNA from FFPE (formalin-fixed, paraffin-embedded) samples can be challenging due to the chemical crosslinking and degradation caused by the fixation process. However, with the right techniques and tools like the Mag-Bind® FFPE DNA/RNA Kit, you can optimize your extraction workflow and maximize nucleic acid recovery from these precious samples. One key technique is ensuring thorough deparaffinization and proper digestion of the tissue to release nucleic acids without further degradation. Since FFPE samples often exhibit crosslinking that hampers extraction, gently reversing these crosslinks through optimized incubation times and temperatures is crucial.
The Mag-Bind® FFPE DNA/RNA Kit employs magnetic bead-based technology which enhances the purity and yield of DNA and RNA by efficiently binding nucleic acids while removing contaminants. Additionally, leveraging automation capabilities compatible with open-ended liquid handlers and magnetic processors can improve consistency and throughput, reducing hands-on time and variability. Still, it’s important to recognize the limitations: overly fixed samples may still pose challenges due to severe degradation, but applying these best practices can help you get the most reliable results possible. By carefully following protocol optimizations and employing the Mag-Bind® FFPE DNA/RNA Kit’s advanced features, researchers can streamline nucleic acid extraction and confidently proceed with downstream analyses.
Conclusions
While extracting nucleic acids from FFPE samples presents unique and sometimes formidable challenges, it is far from an impossible task. By adopting optimized protocols, carefully selecting extraction kits designed for heavily cross-linked samples, and integrating quality control steps, researchers can significantly improve both the yield and integrity of DNA and RNA from these valuable specimens. Advances in extraction technologies and a better understanding of fixation effects now allow us to unlock precious molecular information from archival FFPE tissues that might otherwise have been considered unusable. By maximizing the value of these samples, scientists can enhance the scope and accuracy of molecular research, leading to deeper insights in fields ranging from cancer genomics to retrospective disease studies. Ultimately, overcoming the hurdles of FFPE nucleic acid extraction not only preserves the legacy of existing tissue archives but also propels discovery in biomedical science.