Formalin fixed paraffin embedded (FFPE) biospecimens are a crucial source for biomedical research as they allow extended durations of storage by preserving the morphology and cellular details of the samples. FFPE specimens are very stable and can be stored at room temperature as the proteins are stabilized via cross-linking. In the effort to develop more effective cancer therapies, there is an increasing need for specimens that allow the molecular analysis of tumor tissues and isolation of proteins through a process that is safe, efficient and cost-effective during the deparaffinization process.
Due to increased efforts to develop more effective cancer therapies, there is an increased demand for specimens that allow molecular analysis of tumor tissues and isolation of the proteins. This means that a safe, efficient, reproducible and cost-effective process of deparaffinization is required. Releasing proteins from FFPE specimens can be daunting as the extensive molecular crosslinking that occurs through formalin fixation lowers the efficiency of protein extraction and may also interfere with immunoreactivity. This warrants the development of more advanced and efficient extraction methods.
The following are the three most advanced methods that have been developed and researched regarding their efficiency in extracting protein from FFPE samples.
1. Reverse phase protein arrays (RRPA)
- A study analyzing this method sampled fresh frozen and FFPE specimens from cell lines, xenografts, breast cancer, and renal tissues.
- Specimens were deparaffinized and extracted using 6 different protein extraction protocols.
- SDS-PAGE and Western Blot tests were used to evaluate the yield and level of protein degradation.
- The most efficient protocol allowed efficient extraction of immunoreactive protein from samples and was then used to prepare protein lysates from specimens and subjected to RRPA.
- Reproducibility and Spearman correlation was compared between fresh frozen and FFPE samples.
- 85% of 169 total markers on RPPA showed significant correlation between FFPE and frozen specimens with 23 common markers in all sample sets.
- The FFPE preparations were also able to yield biologically meaningful observations associated with pathway signaling in cell lines and classification of renal tissues.
FFPE specimens can be valuable in generating reproducible and biologically relevant proteomic profiles if optimized protein extraction methods and RPPA with specific marker performance are used according to tissue type.
2. The ConversantBio Method
- This method relied on hot distilled water and only takes 20 minutes.
- This new procedure works on stored FFPE specimens that are up to seven years old.
- Requires an 8μm thickness specimen
- Produces 3 – 4 times as much protein compared to other procedures
- Suitable for routine molecular analysis and western blot analysis
- Studies show success on colorectal, breast, thyroid, ovarian, uterine, kidney, bone, ganglion, stomach, pancreatic, and stomach cancer specimens.
- Tissues sectioned using microtome
- Melt wax and promote tissue adherence by placing paraffin section slides on warming block
- Use 1ml of hot distill water to wash paraffin off the slide to break crosslink matrices.
- Use a clean cell scraper to scrape specimen.
- Specimen transfer to clean 1.5ml microcentrifuge tube
- 200μl lysis buffer and 200μl Laemmli 2x buffer added to the tube
- To preserve protein content and quality, lysis buffer must contain phenylmethylsulfonyl fluoride.
- To prevent protein degradation during storage, add 3% (v/v) β-mercaptoethanol.
- Samples incubated at 100⁰C for 8-10 minutes while 2-3 minutes of intermittent vortexing
- At room temperature, centrifuge at 13,000 rpm for 5 minutes.
- Using clean 1.5ml microcentrifuge tube, collect supernatant.
This method may help identify new biomarkers for specific cancers and tumors due to the efficient extraction of proteins from FFPE specimens. This may lead to proper management and prognosis of diseases. Further studies are needed to research if the extraction procedure can support nucleic acid isolation or proteomic analysis.
3. Protein extraction from over-fixed and long-term formalin fixed tissues
- This study researched the efficiency of protein extraction between an established and new commercial buffer system from FFPE specimens that has been formalin fixed for up to 144 hours and tissue blocks up to 20 years old.
- The new buffer system decreases the protein yield when there is increased fixation time.
- There is a 66% protein recovery using the new buffer system from specimens that are 144 hours compared to specimens fixed only for 6 hours.
- The established (old) extraction procedure recovered less than 50% protein.
- The longer the paraffin blocks were stored, the less efficient the protein extraction.
- The study found that with the new buffer system, 50% more protein could be extracted from FFPE specimens that have been stored for 20 or more years compared to the established buffer system.
- This shows that the new buffer system is superior.
In conclusion, protein extraction from FFPE specimens are increasingly important for various areas of research. New methods such as those mentioned are often needed to overcome the challenges of extraction. Currently, since there is no one method that is suitable for all, more methods should be developed to improve and advance the industry.
PubMed Central - An efficient procedure for protein extraction from formalin-fixed, paraffin-embedded tissues for reverse phase protein arrays
Conversantbio - New Developments in Extracting Protein from FFPE Tissue
PLOS One - Successful Protein Extraction from Over-Fixed and Long-Term Stored Formalin-Fixed Tissues