Rapid dewaxing method for nucleic acid extraction of FFPE sample

A sample and nucleic acid technology, which is applied in the field of rapid dewaxing of nucleic acid extraction from FFPE samples, can solve the problems of toxicity, time-consuming, and personal safety hazards of laboratory personnel in dewaxing reagents, so as to reduce the length of heating steps and simplify dewaxing and lysis steps. , the effect of simplifying the complexity of the operation

Inactive Publication Date: 2018-12-18
SHANGHAI MAG GENE NANOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As the most widely used dewaxing method with the most commercial kits, there are still many problems in the xylene dewaxing method: first, xylene is a toxic organic reagent, which is not good for the health of experimenters; secondly, xylene The dewaxing method requires multi-step reagent addition and centrifugation, the experimental process is cumbersome, and the dewaxing and cracking steps take at least 3 hours
However, after the incubation at 60°C, the sample tube needs to be taken out, and then placed in the heating module after the temperature has risen to 90°C, so as to avoid excessive fragmentation of the nucleic acid due to the long de-crosslinking time caused by the heating process; and under the removed oil layer In the water phase process, problems such as the solidification of the upper oil phase caused by temperature changes can easily lead to problems such as difficulty in taking liquid, inaccurate liquid volume, or the inability to take out the sample solution at all. It takes at least 2 hours, plus the extraction step, the whole experiment takes about 3 hours
[0006] In the existing xylene dewaxing method, the dewaxing reagent is toxic, and a slight misoperation during the experiment will cause harm to the personal safety of the experimenter, and there is a certain risk in use; secondly, it requires multi-step reagent addition and Centrifugal operation, the experimental process is cumbersome and time-consuming
The silicone oil cracking two-step dewaxing method requires at least 2 hours of incubation time, requires two different temperatures, and needs to take out the sample tube during the temperature change process, which increases the overall dewaxing time cost and operational complexity. Not conducive to integration with automation devices

Method used

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  • Rapid dewaxing method for nucleic acid extraction of FFPE sample
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  • Rapid dewaxing method for nucleic acid extraction of FFPE sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Add 200 μL of aqueous phase reagent (10 mM EDTA, 30 mM Tris-HCl, 3M guanidine isothiocyanate, 1% TritonX-100), 20 μL of proteinase K (5 mg / mL) and 50 μL of oil phase reagent (silicone oil) into the FFPE tissue sample In the 1.5mL centrifuge tube 101 of 102, briefly centrifuge after shaking and mixing to obtain the mixed solution 103.

[0038] Put it into the heating device 104 that has been heated to 56°C, and incubate at 56°C for 1 hour. At this time, it can be observed that the mixed solution 103 in the tube is layered, the upper layer is the oil phase layer 105, and the lower layer is the water phase layer 106. After the incubation, take out the lower aqueous phase solution 106 with a pipette gun under heating conditions, or completely remove the lower aqueous phase solution 106 into a new centrifuge tube after cooling to room temperature.

[0039] If necessary, 2 μL RNase A can also be added to the aqueous phase solution, and left at room temperature for 5 minutes t...

Embodiment 2

[0042] Add 150 μL of aqueous phase reagent (1 mM EDTA, 50 mM PBS, 4M guanidine hydrochloride, 1% SDS), 20 μL of proteinase K (20 mg / mL) and 150 μL of paraffin oil into the 1.5 mL centrifuge tube 101 containing FFPE tissue sample 102, shake After mixing, centrifuge briefly to obtain a mixture 103.

[0043] Put the centrifuge tube into the heating device 104 that has been heated to 52°C, and incubate at 52°C for 3 hours. At this time, it can be observed that the mixed solution 103 in the tube is separated, the upper layer is the oil phase layer 105, and the lower layer is the water phase Layer 106. If necessary, the centrifuge tube 101 can be taken out, and the centrifuge tube 101 can be put in after the heating device 104 is heated to 80° C., and incubated for 1 hour. After the incubation, take out the lower aqueous phase solution 106 with a pipette gun under heating conditions, or completely remove the lower aqueous phase solution 106 into a new centrifuge tube after cooling ...

Embodiment 3

[0046] Add 300 μL of aqueous phase reagent (10 mM EDTA, 50 mM Tris-HCl, 1% SDS), 20 μL of proteinase K (20 mg / mL) and 150 μL of xylene into the 1.5 mL centrifuge tube 101 containing FFPE tissue sample 102, shake and mix briefly Centrifuge to obtain the mixed solution 103.

[0047] Put the centrifuge tube into the heating device 104 that has been heated to 56°C, and incubate at 56°C for 2 hours. At this time, it can be observed that the mixed solution 103 in the tube is separated, the upper layer is the oil phase layer 105, and the lower layer is the water phase Layer 106. If necessary, the centrifuge tube 101 can be taken out, and the centrifuge tube 101 can be put in after the heating device 104 is heated to 90° C., and incubated for 30 minutes. After the incubation, take out the lower aqueous phase solution 106 with a pipette gun under heating conditions, or completely remove the lower aqueous phase solution 106 into a new centrifuge tube after cooling to room temperature. ...

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Abstract

The invention discloses a rapid dewaxing method for nucleic acid extraction of an FFPE sample, and relates to the field of nucleic acid extraction of paraffin samples. The FFPE extraction method realizes simple and rapid dewaxing of the FFPE sample by adopting an oil phase reagent capable of dissolving paraffin and an aqueous phase reagent capable of lysing a tissue sample by single-temperature incubation, incubation is carried out at a specific condition for 30 min to 2 h after above reagents are added into a sample tube with FFPE, a lower aqueous layer liquid is taken out by a mechanical device or a pipetting device, and the aqueous layer liquid can be used for subsequent nucleic acid extraction. The invention also provides a rapid dewaxing kit using the method. Dewaxing and tissue cracking operations are carried out at the single temperature, so the experimental flow is simplified, and the overall dewaxing time is shortened; and a specific reagent system is adopted, so ideal dewaxing and nucleic acid extraction effects are achieved.

Description

technical field [0001] The invention relates to extracting nucleic acid from paraffin-embedded samples, in particular to a rapid dewaxing method for extracting nucleic acid from FFPE samples. Background technique [0002] Formalin-Fixed and Paraffin-Embedded (FFPE) is a tissue preservation technique. In order to maintain the nuclear protein structure of this technique, the tissue is first fixed with formalin and then embedded in solid paraffin for histological diagnosis or research after sectioning. [0003] If you want to conduct molecular biology research on FFPE tissue, the first step is to extract DNA. However, the cross-linking effect of the fixative will damage the nucleic acid structure, and the formalin tissue fixation and paraffin embedding will hinder the extraction of nucleic acid, inhibit the activity of DNA polymerase and affect PCR amplification. In addition, paraffin hinders the penetration of digestive juice into the tissue, thereby inhibiting the contact b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 方晓霞徐宏古宏晨
Owner SHANGHAI MAG GENE NANOTECH CO LTD
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