Extracting Nucleic Acids from FFPE Tissues

Preserving specimens as formalin-fixed paraffin-embedded (FFPE) tissue samples is a common method of saving crucial biological information in a timely fashion. It safeguards precious samples, even allowing archival storage, while buying scientists time to study them properly and carefully, with as much attention to detail as needed. However, as the method was not designed with nucleic acids in mind, extracting them from FFPE tissues can be challenging. This article discusses methods of extracting DNA and RNA from FFPE tissues, and factors to consider when selecting an extraction method or kit.

Tried-and-true extraction methods

Formalin (also known as formaldehyde) preserves tissues in part by cross-linking proteins and nucleic acids; this is so effective that fixed tissues can be stored at room temperature. Unfortunately, this also degrades nucleic acids, so extracting DNA and RNA from these samples has required creative adaptations. The removal of paraffin wax can be accomplished with volatile organic solvents, but today non-toxic agents are also available. After wax removal, incubation with protein-digesting enzymes, heat, and other reagents help to free the nucleic acids, which can be purified using spin columns or particle-based methods such as magnetic beads (the latter being more amenable to automation).

Promega offers tools for both column- and particle-based methods. Paraj Mandrekar, Technical Services Scientist at Promega, says that incubating longer during extraction, and adding heat, can help to release nucleic acids—especially longer fragments—from crosslinking to proteins and each other. “Our methods for extracting nucleic acids from FFPE samples normally incorporate a heated incubation as part of the pre-processing, coupled with incubation in proteinase K and a buffer with detergents,” says Mandrekar. “If a user needs longer nucleic acids (for sequencing or to generate longer PCR amplicons, for example), they should try to reduce the fixation time to result in longer intact nucleic acid.”

Newer approaches

Newer approaches add unique twists to the conventional extraction routine. The Arima Genomics HiC+ FFPE kit can extract nucleic acids from new and archival FFPE tissues for 3D genomic analysis, in combination with Illumina's next-generation sequencing instrumentation. “Arima Genomics’ tools optimize extraction using proprietary proximity ligation (Hi-C) chemistry to retain the 3-dimensional chromatin conformation,” says Ibrahim Jivanjee, Arima's Director of Product Management. “These data improve tissue classification and characterization through structural variant detection and can yield insights into gene regulation.” For example, Arima’s Hi-C technology can detect structural variants, including gene fusions and translocations. “Gene fusions as biomarkers have broad clinical utility in cancer patients—including for accurate diagnosis, early detection, prognosis, and selection of optimal treatment regimens,” notes Jivanjee.

Another new approach harnesses the power of sound. Covaris’ Adaptive Focused Acoustics^® (AFA^®) Technology uses acoustic energy to emulsify and remove paraffin, efficiently extracting tissues embedded in FFPE and releasing DNA and RNA from the tissues. The truXTRAC FFPE Total NA Auto 96 kit and workflow allow researchers to extract total nucleic acid (DNA and RNA) from FFPE tissues, without using any harmful organic solvents for paraffin removal. The kit not only ensures high quality and yield of DNA, but also enables isolation and purification of high-quality, amplifiable RNA in a high-throughput workflow environment.

Extraction kits typically used with FFPE samples focus on the extraction and isolation of DNA, and extracting RNA requires an entirely separate extraction workflow. Covaris’ truXTRAC FFPE Total NA Auto 96 workflow allows extraction of both DNA and RNA simultaneously. This can yield better information faster, for tumor studies in cancer research. “[Extracting] DNA alone might not offer a comprehensive picture of the tumor and the disease,” says Deb Bhattacharyya, Senior Director of Marketing at Covaris. “Robust DNA and RNA extraction protocols are required for accurate identification and quantification of somatic variants in FFPE tissue specimens.”

Factors to consider when choosing a method

Several factors are important when choosing how to extract nucleic acids from FFPE tissues, or what type of kit to purchase. Jivanjee recommends considering the sample availability and the type of analysis you want to do when selecting an extraction kit, and how those data will help you answer your biological research questions. “Arima Hi-C technology is a powerful tool for investigating the sequence, structure, and regulatory landscape in cancer and other sample types,” says Jivanjee. “With this technology you can detect structural variants, including gene fusions that have been missed by other approaches, even in FFPE samples archived for 10+ years.”

Choose a method/kit that is compatible with your lab and sample.

Some preprocessing steps may not be advisable for analysis of FFPE samples. For example, some methods remove paraffin by treating tissues with organic solvents such as xylene or citrulline. “Working with xylene and its derivatives can pose some significant challenges,” says Bhattacharyya. “They are hazardous, often requiring a fume hood, and can be difficult when it comes to automation.”

Choose an appropriate quantitation method.

Your quantitation method will need to be appropriate for your sample type, and compatible with your lab and workflow. “Quantitation methods can produce substantial differences in estimated yield, and those estimates may not be useful for subsequent analysis steps,” says Mandrekar. “Depending upon the degree of DNA degradation and desired amplicon length, spectrophotometric methods of quantitation, for example, can provide estimates of yield that are several orders of magnitude higher than quantitation that relies upon generation of a PCR amplicon, such as qPCR-based estimation of yield.”

Choose a throughput level that makes sense for your lab.

Addressing throughput requirements can be tricky for FFPE samples, because often one must accommodate a wide range of throughputs. “From few to thousands of samples—the analytical workflow from extraction to analysis of DNA and RNA must be done with equal efficiency, regardless of sample complexity and user expertise,” says Bhattacharyya. “While it is important to extract and analyze DNA and RNA with confidence from every single FFPE sample, doing so in the shortest possible time offers additional advantage—such as ability to address more samples in the same time.”

Manual nucleic acid extraction is certainly feasible on occasion, but performing them routinely for, say, 10s or 100s of FFPE samples, may warrant consideration of automation tools to free up scientists’ time. “In many cases, isolation of larger numbers of samples is more important than maximizing the yield from each isolate,” says Mandrekar. “Promega offers FFPE nucleic acid purification chemistry based on paramagnetic particle-based isolation that can be implemented on Maxwell instrumentation or high-throughput automation.”

If an extraction technique just right for your most important FFPE tissue samples hasn’t yet emerged, check back often—it may not be long before it appears, as the field quickly evolves.