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Automatic extraction method for nucleic acid in FFPE ((Formalin-Fixed and Parrffin-Embedded) sample

A technology for samples and nucleic acids, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve problems such as inseparability and inability to realize full automation, and achieve easy operation, convenient operation, automatic extraction and easy automation Effect

Pending Publication Date: 2018-11-20
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is inseparable from centrifuge-assisted
And individual steps need to be manually operated, and cannot be fully automated

Method used

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  • Automatic extraction method for nucleic acid in FFPE ((Formalin-Fixed and Parrffin-Embedded) sample
  • Automatic extraction method for nucleic acid in FFPE ((Formalin-Fixed and Parrffin-Embedded) sample
  • Automatic extraction method for nucleic acid in FFPE ((Formalin-Fixed and Parrffin-Embedded) sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 DNA extraction from FFPE samples

[0043] 1. Take 2 FFPE samples of lung cancer into a centrifuge tube and add 1000 μL of xylene;

[0044] 2. Dewaxing by heating in a metal bath at 50°C for 5 minutes;

[0045] 3. Add 100 μL of magnetic beads;

[0046] 4. After adding the magnetic beads, mix well at room temperature for 1 min to fully contact the magnetic beads with the cell tissue in the dewaxing agent. Put the centrifuge tube on the magnetic stand or put a magnetic bar in the centrifuge tube. Stand still for 5 minutes until the magnetic beads are completely transferred to the magnetic stand, and remove all the dewaxing agent with a pipetting device;

[0047] 5. Add 1mL absolute ethanol to the centrifuge tube. After adding the organic solvent, mix well at room temperature for 1 min, and put the centrifuge tube on the magnetic stand. Let it stand for 5 minutes, and when the magnetic beads are completely transferred to the magnetic stand, remove all the org...

Embodiment 2

[0075] Example 2 RNA extraction from FFPE samples

[0076] 1. Take 2 FFPE samples of intestinal cancer into a centrifuge tube and add 1000 μL of xylene;

[0077] 2. Dewaxing by heating in a metal bath at 50°C for 5 minutes;

[0078] 3. Add 100 μL of magnetic beads;

[0079] 4. After adding the magnetic beads, mix well for 5 minutes to make the magnetic beads fully contact with the cell tissue in the dewaxing agent;

[0080] 5. Put the centrifuge tube on the magnetic stand. Let it stand for another 5 minutes until the magnetic beads are completely transferred to the magnetic stand, and remove all the dewaxing agent with a pipetting device;

[0081] 6. Add 1mL of absolute ethanol to the centrifuge tube, after adding the organic solvent, mix well at room temperature for 1min, put the centrifuge tube on a magnetic stand or put a magnetic bar in the centrifuge tube. Let it stand for 5 minutes, and when the magnetic beads are completely transferred to the magnetic stand, remove ...

Embodiment 3

[0093] Example 3 Simultaneous extraction of DNA and RNA from FFPE samples

[0094] After applying 2 deparaffinization and tissue cell rehydration. Add 25 μL proteinase K and 200 μL lysate RTL into the centrifuge tube, place it in a 60°C metal bath and heat for 15 minutes. Since RNA mainly exists in the intercellular substance, DNA mainly exists in the nucleus. Put the centrifuge tube on the magnetic stand and let it stand for 5 minutes until the magnetic beads are completely transferred to the magnetic stand. Use a pipetting device to transfer the digested lysate containing 180 μL RNA to a new centrifuge tube. Complete RNA extraction using the nucleic acid binding, washing, and elution steps mentioned in Application 2. Then add 15 μL of proteinase K and 140 μL of lysate DTL to the magnetic beads adsorbed on the cell tissue in the centrifuge tube, and heat in a metal bath at 60°C for 1-2 hours to completely lyse the cell tissue and release the DNA in the solution. Complete D...

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Abstract

The invention discloses an automatic extraction method for nucleic acid in an FFPE (Formalin-Fixed and Parrffin-Embedded) sample. When DNA or RNA of FFPE is independently extracted, cell tissue can beconfined in a certain space range by magnetic beads through a magnetic separation device, the consequence that tissue is sucked off along with a dewaxing agent or other solution can be effectively avoided, and furthermore the DNA or RNA of FFPE can be extracted; in co-extraction of the DNA or RNA of FFPE, the tissue is dewaxed and rehydrated along with the magnetic beads, lysate RTL is added, andthen RNA can be released into the solution. A centrifugal tube is placed in the magnetic separation device, digestion lysate with the RNA is transferred into a new centrifugal tube by using a liquidtransferring device, and then RNA extraction can be carried out; lysate DTL is continuously put into the magnetic beads into which the cell tissue is adsorbed in the original centrifugal tube, then the DNA can be released into the solution, and DNA extraction can be carried out.

Description

technical field [0001] The invention relates to an automatic extraction method for nucleic acid in FFPE samples. Background technique [0002] The Formalin-Fixed and Parrffin-Embedded (FFPE) method is widely used in the preservation of biological tissue samples, and the vast majority of tissue samples in the world are processed using the FFPE method. With the continuous development of molecular diagnostic technology, more and more molecular biological research and clinical diagnosis rely on the use of FFPE samples as material sources. Use FFPE samples to extract sample DNA and RNA, and then apply it to PCR, NGS and other downstream applications based on nucleic acid analysis. [0003] However, it is quite difficult to extract the nucleic acid from FFPE samples. Because during the preparation of FFPE samples, the tissue has been completely dehydrated and embedded in paraffin solids; while nucleic acid extraction must be in a solution environment, various types of enzymes an...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2527/125
Inventor 林喜建宋庆涛钟书淳廖乃淞郑立谋
Owner AMOY DIAGNOSTICS CO LTD
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