The Process of FFPE Sample Preparation and Nucleic Acid Extraction

FFPE (Formalin-fixed paraffin-embedding) samples are common biomaterials in the medical field. These samples have been widely used in high-throughput sequencing, in situ hybridization, immunohistochemistry and other research fields. Currently, it is estimated that billions of FFPE samples are kept in hospitals or tissue sample banks worldwide, and these tissue samples are one of the most valuable biological resources for disease diagnosis and scientific research.

With the rapid development of genomic testing technologies, especially next-generation sequencing (NGS), which provides unprecedented detection capabilities at the cellular molecular level, researchers can use this technology to study FFPE samples for disease-associated mutations. Let's learn more about FFPE samples from preparation to detection and analysis, and what to look for.

The process of FFPE sample preparation and nucleic acid extraction is as follows.

Sample collection

The procedure to obtain tissue specimens from a patient involves anesthesia, ligation of blood vessels, excision of the tissue, and fixation. During the time between sampling and tissue fixation, gene expression in the tissue may change and tissue autolysis may occur; therefore, the operation should be performed for as short a time as possible before tissue fixation to avoid significant changes in nucleic acid and protein expression profiles.

Formalin fixation

Formalin solution is used for fixation of tissues. For optimal fixation results, a neutral formalin buffered solution should be used instead of an unbuffered or acidic solution. A certain degree of cross-linking between nucleic acids, between proteins, and between nucleic acids and proteins occurs when tissues are fixed in a certain concentration of formalin solution, and the longer the fixation time, the greater the degree of cross-linking.

Paraffin Embedding

After formalin fixation, the tissue needs to be embedded in paraffin, a process that involves dehydration, transparency, wax immersion and embedding. The first step is dehydration, in which water is replaced with an alcohol, usually ethanol. This is followed by clearing, where the alcohol is replaced with xylene or a xylene substitute, followed by wax immersion, where xylene is replaced with paraffin. The final step is embedding, where the entire specimen is surrounded by paraffin. Prior to wax immersion, the tissue specimen must be completely dehydrated, as residual moisture may cause degradation of the sample. Paraffin embedding is a critical step in maintaining protein integrity, as residual moisture may lead to protein hydrolysis.

After fixation and embedding, FFPE samples should ideally be stored at optimal temperature, which slows down the degradation of nucleic acids and proteins. It has been shown that RNA remains largely intact after 1 year when FFPE samples are stored at 4°C, rather than room temperature or higher.

The quality of nucleic acids and proteins extracted from FFPE samples depends on how the samples are treated before, during and after fixation and embedding. Samples that are not treated properly can result in severe fragmentation of nucleic acids, and we need to QC the treated samples for subsequent downstream applications such as nucleic acid extraction and sequencing.

Sample sectioning

Paraffin samples need to be sectioned before extraction, generally 5-6 sections are needed, one microscopic examination to determine the tumor cell content, the other for subsequent extraction processing, the thinness of the section will have some influence on the subsequent observation and extraction.

Dewaxing and hydration

Because of the specificity of FFPE samples, paraffin wax can impede the penetration of the digesting solution into the tissue, thereby inhibiting the contact between proteinase K and the proteins in the tissue and affecting tissue digestion and nucleic acid release. To eliminate the adverse effects of paraffin on DNA extraction and PCR amplification, tissues must be thoroughly dewaxed.

Proteinase K Digestion

In order to separate DNA from bound proteins, FFPE samples are subjected to high temperature and Proteinase K digestion. Proteinase K digests the protein fractions as well as any nucleases that may be present in the sample, and this step also prevents DNA degradation. Care should be taken to manage the digestion time, which can be adjusted according to tissue type and tissue size to allow for adequate DNA release.

Genomic DNA purification

Nucleic acids and proteins in FFPE samples are tightly cross-linked, and after performing tissue digestion the samples have become hydrated, at which point they need to be shaken to uncross-link and release the DNA for subsequent purification. If possible, chemical modifications due to cross-linking should also be reversed, as chemically modified DNA cannot be efficiently recovered during purification and is a poor substrate for PCR or other enzymatic analyses. Care must be taken when breaking cross-linkages and reversing chemical modifications, as the violent reaction conditions may lead to further fragmentation of DNA.

Nucleic acid extraction

Depending on the purpose of the study, the appropriate method is chosen to extract DNA, commonly used are phenol-chloro/form extraction, column membrane method, magnetic bead method, etc.

The DNA quality of FFPE samples can significantly affect the success rate of subsequent fluorescent quantitative or second-generation sequencing library construction and sequencing data metrics. Therefore, extracting high quality DNA from FFPE samples is the key to ensure the success of subsequent experiments.