Views: 0 Author: BunnyTeeth Technology Inc. Publish Time: 2022-04-20 Origin: BunnyTeeth Technology Inc.
In pathological research and analysis, isolated tissues tend to lose their normal structure and function. Therefore, tissue samples are mostly preserved in the form of FFPE samples. These samples provide a valuable resource for retrospective studies to elucidate disease mechanisms, identify therapeutic targets and indicate prognosis.
FFPE samples are generally fixed in formalin and then the tissue is embedded in solid paraffin for ease of transport and long-term storage. This step is critical because the genome and transcriptome in the tissue may change and tissue autolysis may occur between the time the patient's tissue is obtained and the time it is fixed. Therefore, fixation and paraffin embedding should be performed as soon as possible to maintain the integrity of the biomolecules.
The morphological and structural characteristics of the tissue of FFPE samples are observed experimentally or analyzed by immunohistochemistry with antibodies. However, with the development of technology, more and more people tend to choose to apply the latest techniques at the molecular level for in-depth data mining.
PART01 Difficulties and challenges
Paraffin-embedded medical samples are very precious, and the total amount of each sample is limited and irreplaceable. Formalin fixation tends to degrade nucleic acids in tissues to different degrees and cross-link between molecules, and the high temperature infiltration process of paraffin further accelerates the degradation of nucleic acids. Therefore, the challenge for FFPE samples at this stage is the low yield, low integrity and poor quality of extracted nucleic acids, which are difficult to be compatible with downstream qPCR, sequencing and other applications. Today, we would like to summarize some exclusive FFPE nucleic acid extraction secrets to help you conquer FFPE samples.
PART02 Operation secrets
1. Method of dewaxing (thorough dewaxing)
Please be sure to perform thorough dewaxing! Due to the special nature of FFPE samples, there is more dewaxing process than fresh tissue when processing the samples. Different dewaxing methods have an important impact on the quality of the product. Many researchers nowadays use organic solvents such as xylene and mineral oil for dewaxing. If this step is not thoroughly dewaxed, the paraffin wax will prevent the penetration of the digesting solution into the tissue in the next step, thus inhibiting the contact between proteinase K and the proteins in the tissue and affecting tissue digestion and DNA release.
2 Proteinase K tissue 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. Make sure to optimize the digestion time! Too short a digestion time results in insufficient release of DNA, leading to low yields of nucleic acid extraction. Use different digestion times for different tissue types, tissue sizes, and repeat or incubate overnight if necessary.
As mentioned earlier in the FFPE sample preparation, formalin fixation tends to cross-link nucleic acids and proteins. After performing a tissue digestion, the sample has become hydrated. At this point it is necessary to promote the separation of the nucleic acid from the tightly cross-linked proteins and release the DNA in mild an environment. It is recommended to incubate the sample for one hour at 80°C for the purpose of reversing the cross-linking.
4. Extraction and quality control of nucleic acids
There are several well-established methods for nucleic acid extraction, including phenol-chloroform extraction, spin column method, magnetic bead method and so on. You only need to choose according to your experimental needs. Please make sure to QC the extracted products. Most people extract FFPE nucleic acids for clear application purposes, such as downstream microarray chips, NGS sequencing, Real-Time PCR and other experiments to analyze the molecular mechanism. Therefore, not only the yield of nucleic acid should be high, but also the quality such as purity and integrity are key concerns. It is especially recommended to perform quality control after extraction of nucleic acids and to monitor the whole process during the condition-finding phase to improve the success rate of experiments.
PART03 How to perform quality control
1. Concentration: simple concentration measurement is done by ultra-micro-UV spectrophotometer, or by labeling double-stranded DNA with saturated dyes and quantifying double-stranded DNA concentration by fluorophotometer.
2. Quantitative PCR: this is the better way to determine the overall quality of a nucleic acid sample. Information on nucleic acid quantification and integrity is obtained from the amplifiable DNA.
3. Library preparation and sequencing: to improve sequencing coverage and homogeneity.
On the issue of FFPE nucleic acid extraction, the Dana Farber Cancer Institute, affiliated with Harvard University, highly recommended protocols back at the 2014 AACR Annual Meeting to help improve DNA yield and quality.
*This report concludes that the FFPE DNA extraction method is very effective, with a six-fold higher DNA yield than other methods. The extracted DNA products can be efficiently PCR amplified. The coverage of DNA fragments with high GC content and the complexity of sequencing libraries are very high.