What is FFPE
Formalin-Fixed and Parrffin-Embedded (FFPE) is a tissue preservation technique. In order to maintain the nuclear protein structure, this technique is first fixed with formalin and then embedded in solid paraffin for sectioning for histological diagnosis or research.
Application of FFPE technology
Formalin is superior to other fixatives in maintaining tissue cell structure integrity, antigen measurability, and tissue permeability because it can irreversibly crosslink with protein amino groups. Therefore, FFPE technology has been widely used in clinical testing and medical science research for a long time, especially cell morphology research and tumor pathology research. With the continuous development of molecular diagnostics in recent years, FFPE technology is increasingly being used for molecular diagnosis of tumor diseases.
Why dewaxing?
If you want to conduct molecular biology research on FFPE tissue, the first thing is to extract DNA. However, the cross-linking action of the fixative is harmful to the nucleic acid. Formalin paraffin and cross-linking hinder the extraction of the nucleic acid and inhibit the activity of the DNA polymerase to affect the PCR amplification. In addition, paraffin blocks the penetration of the digestive juice into the tissue, thereby inhibiting the contact of proteinase K with intracellular proteins, affecting tissue digestion and DNA release. Therefore, in order to eliminate the adverse effects of paraffin on DNA extraction and PCR amplification, it is necessary to completely dewax the tissue without ensuring the increase of exogenous PCR inhibitors and minimizing the additional damage of DNA.
Common processes and problems of xylene dewaxing
The dewaxing method currently applied in the mature kit is mainly an organic solvent dewaxing method, and the xylene dewaxing method is most widely used in tumor research due to its thorough dewaxing characteristics.
In the xylene dewaxing method, the paraffin-embedded structure needs to be immersed in a xylene solution for dewaxing, followed by two high-temperature treatments, one for each hour, at 56 ° C and 90 ° C, respectively. During this time, the digestion buffer and proteinase K were combined to digest the tissue, while gradient ethanol was used to loosen the tissue structure, promote digestion of the digestive juice, and remove trace amounts of xylene and formaldehyde from the tissue.
Figure 1 Basic steps of the xylene dewaxing process
Although the xylene dewaxing method is currently the most popular dewaxing method and is used in most commercial kits, it still has certain problems: First, xylene is a toxic organic reagent during the experimental process. If the body is exposed to a certain degree of harm, this experiment has certain risks; secondly, the experimental process of the dewaxing method is very cumbersome, and the dewaxing step takes at least 3 hours or more, plus extraction The steps of the whole experiment are about 4 hours, and there are many types of reagents involved, which are prone to errors. Therefore, companies are actively developing more efficient, safe and simple solutions to replace xylene dewaxing.
Improvements and advantages of one-step dewaxing
In order to simplify the dewaxing process of paraffin-embedded tissues, a new one-step dewaxing method was developed. The specific steps are as shown in the figure below. The new one-step paraffin lysate (non-toxic) and tissue lysate are added to the centrifuge tube and mixed with the sliced ​​paraffin tissue sample, and incubated at 60 ° C and 90 ° C for 1 hour. The liquid is divided into two layers, an oil layer and an aqueous layer, to complete dewaxing, and the denucleated nucleic acid is present in the aqueous layer.
Figure 2 basic steps of one-step dewaxing
Compared with the traditional xylene dewaxing method, the advantages of this new one-step dewaxing method are mainly reflected in the following aspects:
Table 1 Difference between traditional xylene dewaxing method and one-step dewaxing method
project | Traditional xylene dewaxing | One-step dewaxing |
Paraffin solution type | Toxic substance xylene | Use non-toxic solution |
Separation method | Centrifuge centrifuge is required to remove the supernatant | Oil moisture layer, no additional operation required |
Dewaxing cracking connection | Dewaxing---air drying---adding lysate to lyse tissue | Dewaxing and cracking simultaneous heating, saving steps |
Automation compatibility | Uncompatible automation | Due to its one-step nature, no additional manual operation is required, ideal for integration into automated instruments |
waste time | 3 hours | 2 hours |
In the DNA extraction effect, the following experiment compares the extraction yields of large, medium, and small samples by conventional dewaxing and one-step dewaxing.
Large sample : Samples with an area between 2.5-4 mm 2 , a thickness of 5-20 um, and a volume between 0.0125-0.08 mm 3 after sectioning.
Medium sample : Samples with an area between 1.5-2.5 mm 2 and a thickness of 5-20 um and a volume between 0.0075-0.05 mm 3 after sectioning.
Small sample : Samples with an area between 0.2-1.5 mm 2 and a thickness of 5-20 um and a volume between 0.001-0.03 mm 3 after sectioning.
Figure 3: The difference between the traditional dewaxing method and the one-step dewaxing method in the extraction yield
The results show that in a large number of samples, the extraction yields of the two methods are comparable; for medium and small samples, the yield of one-step dewaxing is greater than the traditional dewaxing method. Therefore, the one-step dewaxing method not only simplifies the operation, reduces the risk, but also improves the extraction efficiency.
How to integrate automation solutions with one-step dewaxing
Among the above advantages, the decisive advantage of the one-step dewaxing method over the conventional xylene dewaxing method is the compatibility with automation. The one-step method makes it very simple to integrate into the magnetic bead automated nucleic acid extractor. Reduce the steps that require manual involvement. Only need to put the sliced ​​sample into the pre-installed reagent tank, prepare the required consumables, wait after starting the operation, free the experimenter time, and greatly improve the experiment efficiency.
Now, TIANGEN has developed a one-step DNA extraction kit for paraffin tissue using this method. With the TGuide M16/48 magnetic bead automatic nucleic acid extractor, dewaxing, DNA extraction and purification of paraffin section tissue samples can be completed within 3 hours. For details, please see: http://?productShow/t1/10/id/334.htmlhttp://?productShow/t1/10/id/334.html
references:
[1] Tan Zhuoyi, Ding Mei, Wang Baojie. DNA extraction from formalin-fixed paraffin-embedded tissues. Forensic Medicine, 2006(6): 455-458
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What is the application of CRL-40 941?
* Applied in the pharmaceutical field.
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