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HomeBrancheninformationenBasic knowledge: formalin-fixed paraffin-embedded tissue DNA extraction

Basic knowledge: formalin-fixed paraffin-embedded tissue DNA extraction


Basic knowledge: formalin-fixed paraffin-embedded tissue DNA extraction

Source: Shenzhen An Bi Sheng Technology Co., Ltd. Reprinted please indicate the source [size: large, medium and small]

This article is about a topic that we rarely ask questions, because it's too simple, and the method is very routine and rarely makes mistakes. What I want to say is: DNA extraction of formalin-fixed paraffin-embedded tissue.

What is an FFPE organization?

FFPE refers to a tissue sample (usually suspected tumor tissue) that is fixed in formalin and then embedded in paraffin in order to maintain a nuclear protein structure for cutting into 5-10 micron thick slices using an ultramicrotome. Formalin and the protein amino group undergo irreversible cross-linking to protect the structural integrity of the cell, and staining shows the deformed structure caused by the tumor in the tissue. However, the crosslinking of the fixative is detrimental to the nucleic acid. Formalin paraffin and cross-linking also prevent the extraction of nucleic acids, inhibition of deoxyribonucleic acid polymerase, PCR amplification, must be removed.

The prospect of obtaining DNA from these samples appears to be bleak. Fortunately, we have already served these two main obstacles, and it is no longer difficult to obtain high quality DNA from FFPE organizations.

How to remove paraffin?

The traditional method of removing paraffin is usually to use xylene, a highly flammable organic solvent. The tissue sections were washed several times with xylene to dissolve the paraffin, and then washed repeatedly with ethanol to remove residual xylene before DNA extraction. This repeated number of rinsing steps, each time a small amount of tissue is washed away with paraffin and organic solvents.

Another method is to use the non-toxic BiOstic Paraffin Removal Reagent (Cat. No. 12251-50). It is as good as xylene and safe and biodegradable.

Can you get high yield DNA without going to paraffin?

I am very glad that you have asked this question. Yes, yes. If you have a way to increase the activity of proteinase K so that it can penetrate the paraffin to digest, then you don't have to remove the paraffin first. Reduce processing time and avoid tissue loss.

In order to achieve this effect, we have introduced a pair of solution combinations to create a strong denaturing environment, increase the activity of proteinase K at high temperatures, and complete tissue digestion while paraffin is dissolved. If you have used the BiOstic FFPE Tissue DNA Kit, you know that they are FP1 and FP2 solutions, respectively. The synergistic effect of these two solutions is much higher than the activity of the standard proteinase K digestion buffer, resulting in high yield DNA as shown in the figure below. In this example (provided by a customer), each piece was added a 10 micron thick section removed from the histological slide. The blue sample was pretreated with xylene, the DNA extraction was performed according to the manufacturer's instructions, and the BiOStic sample was directly extracted without the paraffin removal step. The yield was measured spectrophotometrically. As you can see, the yield of each piece of 1-2 micrograms varies from 0.5-10 μg. No single slice results are similar.

What is the molecular weight of DNA?

The molecular weight of DNA is largely influenced by the fixed treatment method and the fixed time. It is also subject to the age limit of the organization itself. Usually the molecular weight is very small, about 100-500 bp. As you can see, by avoiding the use of organic solvents and additional processing steps, further damage and loss of DNA can be reduced (note the M strip in the figure, not treated with xylene). Since DNA can only be known by performing a complete extraction, we generally recommend the use of qPCR assays designed specifically for the smallest amplicon. In the example to the right, the assay is specific to the human GAPDH gene and the amplicon size is 70 bp.

Can I extract RNA from FFPE tissue?

It is clear that the RNA degradation of such samples can be very serious. They are not made in an RNase-free environment, and tissue samples may have been stored in cabinets at room temperature for many years. But we still have ways to extract RNA from FFPE tissue and get valuable data. By slightly modifying several steps, we were able to extract RNA using the BiOstic FFPE DNA Isolation Kit. The combination of FP1 and FP2 is too strong for RNA, so we used a milder neutral solution BF2 for RNA (Cat. No. 24000-50-2). The 90 ° C incubation step to remove protein crosslinks during DNA extraction was not possible. Anas of chemical modification of RNA from formal in-fixed samples and optimization of molecular biology applications for such samples, Nucleic Acids Research, 1999, vol. 27, No. 22, pages 4436- In 4443), the temperature gradient method was used to determine which temperature and how long the RNA was extracted during the extraction process, which could remove both protein cross-linking and RT-PCR. They found that 30 minutes at 70 ° C is enough to do the job.

If you are interested in our FFPE DNA kit or RNA extraction procedure, please let us know. We will give you a trial.

to sum up

The unique sample type of FFPE has many challenges, and we have made the DNA extraction part quite easy.

Recently published references using the BiOstic FFPE DNA Tissue Kit:

Shawn E. Yost, Erin N. Smith, Richard B. Schwab, Lei Bao, HyunChul Jung, Xiaoyun Wang, Emile Voest, John P. Pierce, Karen Messer, Barbara A. Parker, Olivier Harismendy, and Kelly A. Frazer. Of high-confidence somatic mutations in whole genome sequence of formalin-fixed breast cancer specimens. Nucleic Acids Res . 1–12 (2012).