About a century ago, an excellent method of preserving tissue samples was developed. That technique, known as Formalin-Fixed, Paraffin-Embedded (FFPE) tissue, was a step forward for biospecimen research. However, FFPE was developed primarily for histological applications. Today, there is great interest in using FFPE biospecimens for molecular biological research.
Utilizing top-quality, carefully-sourced FFPE tissue is helping move key research protocols forward, with the ultimate goal of finding new and better treatments for many intractable diseases. FFPE biospecimens find wide use for large-scale drug discovery and biomedical research efforts. Important indications include use in biomarker identification and validation, genetic studies, and tissue structure visualization. Such samples are used for studies of a variety of cancer types, including breast, lung, bladder and colon.
A key goal for many researchers using FFPE tissue samples is extraction of high-quality DNA or RNA nucleic acids. However, obtaining good quality, high purity nucleic acids from FFPE tissues is a challenge because of the fixation and embedding process. Formaldehyde causes cross-linking that hooks nucleic acids to each other, as well as to proteins nearby. Since this is a chemical reaction, it is not easily reversed. Further, the paraffin used to embed specimens must be carefully removed; if not, tissue rehydration and crosslink reversals may not be optimized, leading to poorer yields and quality.
Fortunately, industry ingenuity has led to the development of a variety of methods to successfully extract nucleic acids from FFPE samples. There is no one set method for extraction, so it's important for you to identify your ultimate application post-extraction.
Typically, the extraction process begins with the removal of paraffin. This is usually performed in a passive manner using xylene or another type of solvent to dissolve the paraffin. There is also a so-called active technique that utilizes acoustic energy to de-paraffin tissue specimens. Proponents of this method claim it results in superior tissue rehydration and molecule yields.
There are several nucleic acid extraction techniques:
Magnetic bead-based DNA reversible binding method (Beckman Coulter)
After tissues are digested and release their nucleic acids, they are bound to magnetic beads. The beads are separated and the captured products washed with an ethanol solution, then isopropanol to remove contaminants, then once more with ethanol, followed by nucleic acid elution from the magnetic particles. In one objective study of the different methodologies, it was found that this technique improved contamination removal, but reduced the maximum amplifiable fragment length.
Nucleic acids are released from tissue sections using special lysis conditions, then applied to a glass fiber column which is said to immobilize them while contaminants are removed. Again, one recent objective study concluded that column-based methods provided good nucleic acid concentrations and yields, however there were other chemical contaminants present.
Lysis only (Epicentre Biotechnologies)
This method uses salt precipitation for extracting nucleic acids. This technique is claimed to result in higher yields and less sample loss, plus results in one hour. According to the study, this method offers no nucleic acid purification, though large quantities of amplifiable materials are obtained.
Plate-based solid-phase reversible binding technology (Azco)
Using solid surface reversible binding plate technology, the company claims that DNA and RNA are bound to the plate walls and so isolated from other proteins, salts, and debris for easy purification. It is said to be quicker and less expensive than other nucleic acid extraction processes. This method was found in one study to have the lowest nucleic acid yield and concentration and shortest fragments, but also the ability to process many samples at the same time.
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