Biobanks may choose to store biological samples as formalin-fixed paraffin-embedded (FFPE) blocks. FFPE preservation is the standard method of collecting and storing surgical pathology samples. This biobanking method preserves tissue morphology at room temperature for many years and is more cost-effective than cryopreservation1. Biobanked FFPE tissue samples can be used in hundreds of different assays, but are most commonly used for immunohistochemistry and histological studies2.
Formalin Fixation Damages DNA, RNA and Protein
Formalin fixation cross-links macromolecules, making DNA, RNA and protein difficult to extract. However, a number of recent papers have reported success in isolating fragmented DNA, RNA and protein from FFPE blocks. Due to fragmentation, only short segments of DNA up to 250bp can be amplified from FFPE samples3. Despite this limitation, DNA from FFPE samples has been used in PCR and sequence-specific oligonucleotide (SSO) probe assays. RNA also degrades during the formalin fixation process and RNA integrity is much lower in FFPE samples than in frozen samples1.
Macromolecules May Degrade Over Time in Paraffin Blocks
While it is known that DNA, RNA and protein quality is compromised by the formalin fixation process, it isn’t clear whether these macromolecules further degrade in FFPE samples over time. In one study, researchers from the University of Pennsylvania compared the quality and quantity of genomic DNA, total RNA and total protein extracted from their biobanked FFPE blocks of lung, thyroid and salivary gland tumors1. They found no difference in DNA or RNA from blocks biobanked for 11 to 12 years, compared with nucleic acids from new blocks. However, they did find more protein degradation in samples 5 years and older.
The researchers measured DNA and RNA quality and quantity by NanoQuant. DNA from FFPE samples stored for up to 11 to 12 years had statistically similar 260/280 ratios to DNA from new FFPE samples or from frozen samples. However, the RNA Quality Indicator (RQI) was significantly higher in RNA extracted from frozen samples than in RNA from FFPE samples. Older FFPE samples had the same RQI as recently embedded samples – all RQIs were under 3, indicating significant RNA degradation. Protein quantity was measure by BCA assay and protein quality was analyzed by electrophoresis. Protein quantity did not decrease in FFPE samples over time. However, protein extracted from samples 5 years or older showed significantly more fragmentation than younger samples.
miRNA May Be More Stable than mRNA in Paraffin Blocks
A separate study analyzed RNA and microRNA integrity in samples fixed by the non-formalin fixative PAXgene and embedded in paraffin blocks (PFPE)2. PAXgene fixation does not cross-link macromolecules and so theoretically should cause less damage to macromolecules. However published studies show inconsistent results regarding the quality of DNA, RNA and protein extracted from PAXgene-fixed samples. This study measured RNA and miRNA integrity by RNA integrity number (RIN) and reverse transcription polymerase chain reaction (RT-PCR). Results showed that over a 7-year period, mRNA degraded in PFPE blocks whereas miRNA remained stable. This is likely due to the fact that miRNAs are much shorter than mRNAs.
Fixing and embedding tissues in paraffin blocks is a cost-effective method of storing biological samples. These samples can be used for histomorphological and immunohistochemical studies. However, it is not currently possible to extract intact RNA, DNA or protein from fixed tissues. Moreover, there is evidence that macromolecules further degrade in paraffin blocks over time. Therefore, cryopreservation remains the preferred method to maintain the integrity of macromolecules in biobanked samples.
- Kokkat et al. Archived Formalin-Fixed Paraffin-Embedded (FFPE) Blocks: A Valuable Underexploited Resource for Extraction of DNA, RNA and Protein. Biopreserv Biobank. 2013
- Sanchez et al. RNA and microRNA Stability in PAXgene-Fixed Paraffin-Embedded Tissue Blocks After Seven Years’ Storage. Am J Clin Pathol. 2018
- Dedhia et al. Evaluation of DNA extraction methods and real time PCR optimization on formalin-fixed paraffin-embedded tissues. Asian Pac J Cancer Prev. 2007