The archives of formalin-fixed paraffin-embedded (FFPE) tissue sections stored around the world represent a valuable and extensive source of material for biomedical research. With more researchers turning towards molecular analysis of FFPE samples, it is becoming increasingly important to develop specific protocols that take into consideration the unique nature of these samples. The complete FFPE workflow starts with preparation and archiving to minimize the reduction in quality of DNA, RNA and protein caused by fixation and embedding processes. The process ends with retrieval and analysis to efficiently recover analyzable biomolecules from FFPE samples and what necessary modifications need to be made to downstream applications such as reverse transcription and real-time PCR.
The quality of the nucleic acids and proteins extracted from FFPE samples depends on how the samples were handled before, during and after fixation and embedding. When preparing and archiving FFPE samples, it is important to take the following considerations into account, as they influence biomolecule quality:
- • Sample Type/Handling – Each tissue sample is different and this impacts the speed in which biomolecules are degraded, induced or modified following collection. When obtaining a sample, it’s important to note the duration of each step of the procedure as gene expression in the tissue will change over time. Tissue autolysis can also occur between patient anesthetization and tissue fixation.
- • Formalin Fixation – Fixation of tissue involves placing specimens in a formalin solution, which can vary in composition. The resulting chemical reaction leads to crosslinks between biomolecules, including crosslinks between nucleic acids, between proteins, and between nucleic acids and proteins. For optimal results, neutral-buffered formalin solution should be used as it slows down the degradation of formalin, which is believed to contribute to impairment of nucleic acid quality. Also, smaller tissue specimens should be used as the rate of formalin penetration is correlated to the tissue thickness.
- • Paraffin Embedding – After fixation in formalin, tissue specimens are embedded in paraffin, a process which consists of several steps, including dehydration, clearing and embedding. Paraffin embedding is a critical step for the maintenance of protein integrity, as residual water can lead to proteolysis.
- • Staining – To avoid problems in molecular analysis of FFPE samples, sample staining should be avoided as certain stains and reagents may adversely affect nucleic acids, especially those used for immunohistochemical staining. If sample staining is required, the use of cresyl violet stain is preferred, as it is compatible with nucleic acid analyses. Purification of proteins from sections that have been stained using histology stains (e.g., hematoxylin or fast red) can result in dramatically decreased protein yield. However purified protein can be used for downstream analysis such as western blotting or reverse-phase protein microarray analysis.
- • Storage – After fixation and embedding, FFPE samples should ideally be stored at an optimal temperature that slows down the degradation of nucleic acids and proteins.
The FFPE sample spectrum may seem complex from sample extraction to storage, but it is important to maintain sample quality. In the next series of posts, we will look at the critical factors used for retrieval of usable analytes from FFPE samples and the critical factors for successful molecular analysis of FFPE samples.