Method for isolating nucleic acids from formalin-fixed paraffin embedded tissue samples

A paraffin-embedded, formalin technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA preparation, etc., can solve problems such as accelerated equipment wear, unfavorable automation, and interference operations.

Inactive Publication Date: 2014-12-24
RBC BIOSCI CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in practice, this method does not fully dissolve or separate the paraffin used for embedding tissues, and the paraffin begins to solidify near its melting point (usually below 60 degrees), and gradually condenses into a gel or solid state. Paraffin, therefore, the solution in each step of the operation process of this method is relatively sensitive to temperature, that is, a certain temperature must be maintained to avoid the paraffin from condensing; otherwise, the paraffin may condense into a solid state due to the temperature drop and suspend or settle in the solution In, interfering with the operation
Especially, when the user intends to apply this nucleic acid extraction technology to automated equipment, this phenomenon is very unfavorable. The reason is that: since the existing automated nucleic acid extraction equipment is equipped with a large number of pipeline components and pipetting components, it is difficult for the extraction step The solution quality requirements are relatively high, so that the pipeline can run smoothly, and the non-liquid components caused by the paraffin condensation phenomenon that may be caused by the above method will cause pipeline blockage, interfere with the operation of automatic equipment, and in severe cases may speed up the equipment. The loss of the product; and related problems such as the extraction and purification quality of the product, so it is necessary to deal with this loss with additional steps and equipment (such as centrifugal separation, filter or filter screen, etc.), so it is very unfavorable for automation

Method used

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  • Method for isolating nucleic acids from formalin-fixed paraffin embedded tissue samples
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  • Method for isolating nucleic acids from formalin-fixed paraffin embedded tissue samples

Examples

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Effect test

preparation example Construction

[0039] According to the aforementioned FFPE tissue sample preparation principle, if you want to analyze the biological tissue in the FFPE tissue sample, you must first overcome the obstacles of paraffin materials. To solve this problem, the dewaxing method used in the past (please also refer to figure 1 ) Mainly use organic solvents, such as toluene, xylene, etc., to dissolve the paraffin material, and after multiple replacements of the organic solvent, the paraffin can be fully removed; then a series of "oil phase solution "Mixed and replaced with the aqueous solution" complex steps, the use of alcohol solvents (such as ethanol or isopropanol) to wash away the aforementioned organic solvents is called "rinsing"; or further replaced with aqueous solvents, called "rehydration" (Rehydration) step, can obtain the biological tissue for nucleic acid separation and extraction, and the biological tissue may be lost in the complicated operation, which significantly affects the recovery ...

Embodiment 2

[0073] Pretreatment method for dissolving paraffin wax with xylene (pretreatment 1)

[0074] This embodiment lists a preferred embodiment of the present invention. The large, medium, and small FFPE tissue samples are respectively pre-processed, and the paraffin material in the FFPE tissue sample is simultaneously dissolved and the biological tissue sample is lysed. The column separation method" separates and obtains nucleic acid products. The implementation steps are as follows:

[0075] First, add 400μl of xylene (oil phase solution), 180μl of water phase solution (1% SDS; 30mM Tris-Hcl; 10mM EDTA) and 20μl of proteolytic enzyme K (proteinase K) into a microcentrifuge tube containing FFPE tissue samples , Shake and mix for 10 seconds to obtain a mixed solution 2A. After that, the heating step is performed, which is to heat the aforementioned mixture 2A at 56°C for 1 hour, and then let it stand at room temperature. At the same time, the heater can be heated to 90°C. After the hea...

Embodiment 3

[0079] Pretreatment method for dissolving paraffin with 6-bromohexyl acetate (pretreatment 2)

[0080] This embodiment lists a preferred embodiment of the present invention. The large, medium, and small FFPE tissue samples are respectively pre-processed, and the paraffin material in the FFPE tissue sample is simultaneously dissolved and the biological tissue sample is lysed. The column separation method" separates and obtains nucleic acid products. The implementation steps are substantially the same as those in Example 2, and the difference is only that the oil phase solution used in Example 3 is 6-bromohexyl acetate, so the implementation details will not be repeated.

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Abstract

Methods are disclosed for isolating nucleic acids from formalin-fixed paraffin embedded (FFPE) tissue samples. Each of tissue samples contains paraffin and a target biological tissue or material, and the method includes the steps of: adding a first reagent and a second reagent to the FFPE tissue sample, the first reagent dissolving the paraffin material and the second reagent lysing the biological tissue; mixing the first reagent, the second reagent, and the FFPE tissue sample to form a first mixture; (2) heating the first mixture at 50-80 DEG C for 30-90 minutes; and then heating the first mixture at 80-95 DEG C for 30-90 minutes to fractionize the first mixture to form an aqueous phase and an oil phase; (3) collecting an aqueous solution from the aqueous phase; and (4) isolating nucleic acids from the aqueous solution. The method improves the efficiency and convenience of isolating nucleic acids from FFPE tissue samples.

Description

Technical field [0001] The present invention relates to a nucleic acid separation method, in particular to a nucleic acid separation method for obtaining nucleic acid from formalin-fixed paraffin-embedded samples. Background technique [0002] Formalin-fixed paraffin-embedded (FFPE) is currently the most widely used method for preserving pathological samples. Especially for clinical pathological diagnosis and pathological mechanism research, formalin fixed paraffin embedding method and tissue samples are one of the indispensable tools. Since biological tissue samples are easily corrupted and difficult to preserve for a long time after they are in vitro, formalin-fixed paraffin-embedded tissue provides a perfect solution: through proper formalin-fixed paraffin embedding, the sample can fully retain its The original appearance of the tissues can be preserved for long-term preservation; it is also possible to construct a pathological sample database through comprehensive archiving,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q1/6806
Inventor 钟挺豪官振群郭时钰
Owner RBC BIOSCI CORP
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