There are a large number of tissues stored for globally extended durations that need to be formalin fixed and paraffin embedded (FFPE) as it preserves the morphology and cellular details of the samples. The formalin paralyzes cell metabolism while the paraffin seals the tissue and reduce oxidation. At ambient temperatures, it is also more cost effective compared to storing frozen tissues due to the maintenance, space, and labor costs. The FFPE technique provides an important source for retrospective genetic studies, drug discovery, and biotech research. Due to increasing advancements in the industry, routine tests now include the detection of bacteria and viruses, neoplasm-associated mutations, up or down regulation of mRNAs, and neoplasm-associated mutations.archives of FFPE tissues that have been maintained for decades are now an extraordinary source that allow retrospective studies that can be used to correlate molecular findings, therapy, and clinical outcome.
Advantages and Disadvantages of Formalin Fixation
| Advantages | Disadvantages |
|---|---|
| Ease of tissue handling | Induces cross-links, preventing amplification |
| Possibility of long term storage | Nucleic acid fragmentation may occur |
| Optimal histological quality | Previously thought that DNA extraction and downstream processes can be difficult especially in longer stretches of DNA templates |
| Available in large quantities at low prices |
Proteinase K digestion is routinely combined with other commercial extraction methods. Here, the four different extraction protocols compared with or without the use of proteinase K digestion are:
Heat treatment
QIAamp DNA-blood-mini-kit extraction
EasyMAG NucliSens extraction
Gentra Capture0Column-kit extraction
The impact of these methods on downstream molecular techniques were then evaluated.
Factors studied include:
PCR inhibition by monitoring the amplification of the internal control DNA virus
Performance of isolated DNA in SNP analysis using real time PCR
Performance in conventional multiplex PCR amplifying 200, 400, 600 bp human DNA fragments
The size of the DNA fragments to be amplified is highly dependent on the method of extraction. In all Gentra extracts, amplification inhibitors were found.
A study tested human DNA extracts from the 4 methods mentioned above to see if there are any inhibiting substances, and in two downstream applications:
Real time SNP amplification
Multiplexed 200-400-600 bp PCR
This study used relatively fresh specimens since this is the common scenario as samples are usually investigated for diagnostic purposes. The formalin fixative was also buffered. Both these factors (specimen age and buffering capacity) are known factors that can impact nucleic acid fragmentation. The findings were as follows.
Heat treatment
- Suitable for purifying DNA from paraffin embedded tissues but require proteinase K digestion.
- Second best method for amplification of longer DNA fragments of up to 600 bp.
- Advantage of reduced hands on time when extracting samples.
QIAamp DNA-blood-mini-kit extraction
- Commonly used in routine molecular diagnostics in the detection of pathogens, mutation screening in cancer critical genes.
- Require proteinase K digestion.
- This method also performs well in real time SNP detection after proteinase K digestion.
- Proteinase K digestion is needed to purify paraffin embedded DNA and fragments above 200 bp.
- Performed well for real time SNP detection
- Best method for amplification of longer DNA fragments of up to 600 bp.
EasyMAG NucliSens extraction
- Just like the QIAamp method, the EasyMAG is also commonly used in routine molecular diagnostics in the detection of pathogens, mutation screening in cancer critical genes.
- Proteinase K digestion required.
- This method also performs well in real time SNP detection after proteinase K digestion.
- Proteinase K digestion is needed to purify paraffin embedded DNA and fragments above 200 bp.
- Performed well for real time SNP detection.
- Third best method for amplification of longer DNA fragments of up to 600 bp.
- Advantage of reduced hands on time when extracting samples.
Gentra Capture-Column-kit extraction
- This method can be used successfully on blood samples but since there is reduced amplification of PhHV which is the internal control virus, it was found that this method could not sufficiently remove inhibitory substances.
- From the cycle threshold value (Ct) values, it also implies that proteinase K digestion is not needed to remove inhibitory substances that may be present.
- Multiplex PCR results after Gentra extraction were very poor.
- Gentra method do not appear suitable to purify DNA from FFPE tissues possibly due to the inability to remove DNA tissue protein cross-links leading to the loss of DNA during the washing steps of DNA extraction.
In conclusion, extraction methods greatly influence the downstream molecular analysis. Of the four methods that were tested:
- The Gentra method was found to be unsuitable for DNA recovery from FFPE tissues
- QIAamp and EasyMAG methods are best for real time SNP detection
- The methods QIAamp followed by heat treatment and EasyMAG are most successful for amplification of 400-600 bp fragments.
Hence, the method of choice used to isolate DNA from FFPE tissues should match the purpose of application.
References
