Automated Nucleic Acid Extraction Method from FFPE Samples

Abstract

This invention discloses an automated method for nucleic acid extraction from FFPE samples. When extracting FFPE DNA or RNA alone, magnetic beads, using a magnetic separation device, confine cells and tissues within a specific space, effectively preventing tissue from being drawn away along with the dewaxing agent or other solutions, thus facilitating the extraction of FFPE DNA or RNA. When co-extracting FFPE DNA and RNA, after dewaxing and rehydration with the magnetic beads, lysis buffer RTL is added to release RNA into the solution. Centrifuge tubes are placed on the magnetic separation device, and the RNA-containing digestion lysis buffer is transferred to a new centrifuge tube using a pipette for RNA extraction. Meanwhile, the magnetic beads containing cells and tissues in the original centrifuge tube are further treated with lysis buffer DTL to release DNA into the solution, allowing for DNA extraction.

Description

Technical Field

[0001] This invention relates to an automated method for extracting nucleic acids from FFPE samples. Background Technology

[0002] Formalin-fixed and paraffin-embedded (FFPE) methods are widely used for the preservation of biological tissue samples; the vast majority of tissue samples worldwide are processed using FFPE. With the continuous development of molecular diagnostic technologies, an increasing number of molecular biological research and clinical diagnostics rely on FFPE samples as a material source. DNA and RNA are extracted from FFPE samples and then applied to downstream nucleic acid-based analysis applications such as PCR and NGS.

[0003] However, extracting nucleic acids from FFPE samples is quite challenging. This is because during FFPE sample preparation, the tissue is completely dehydrated and embedded in paraffin solid; while nucleic acid extraction requires a solution environment for various enzymes and compounds to function. Therefore, generally speaking, to extract nucleic acids from FFPE samples, dewaxing must be performed first to restore the bound water in the tissue cells, creating a foundation for nucleic acid extraction.

[0004] Traditional FFPE nucleic acid extraction methods mainly involve dewaxing with xylene or xylene substitutes, rehydrating the tissue with ethanol solution, digesting and decrosslinking the tissue with protease, and finally extracting nucleic acids using a silica gel mold column or magnetic bead method.

[0005] FFPE magnetic bead method Applied Biosystems TM MagMAX TM The FFPE DNA / RNA Ultra Kit can isolate nucleic acids from FFPE samples. It allows for the sequential extraction of DNA and RNA from the same sample, enabling various sample analyses. However, this method requires a centrifuge and some steps need to be performed manually, so it cannot be fully automated. Summary of the Invention

[0006] The main objective of this invention is to provide a method for extracting nucleic acids from FFPE samples.

[0007] The technical solution adopted by this invention to solve its technical problem is:

[0008] Technical Solution 1:

[0009] The method for extracting nucleic acids from FFPE, wherein the extraction of nucleic acids is to extract DNA or RNA from FFPE separately, firstly, a dewaxing agent is added to the FFPE sample, and after the sample is dewaxed, magnetic beads are added to ensure that the magnetic beads are in full contact with the cells and tissues in the dewaxing agent. Then, a magnetic separation device is used to confine the cells and tissues within a certain space, and then the dewaxing agent is removed to prevent the tissues from being drawn away along with the dewaxing agent or other solutions, thereby extracting DNA or RNA from the FFPE.

[0010] Technical Solution Two:

[0011] A method for extracting nucleic acids from FFPE, wherein the extraction of nucleic acids involves the simultaneous extraction of DNA and RNA from FFPE, firstly, a dewaxing agent is added to the FFPE sample. After dewaxing, magnetic beads, proteinase K, and lysis buffer RTL are added to a centrifuge tube. By controlling the temperature and time, the cell membrane is ruptured while the nuclear membrane, cytoskeleton, and other structures remain intact, allowing RNA to be released into the solution. The centrifuge tube is then placed on a magnetic separation device, and the digestion lysis buffer containing RNA is transferred to a new centrifuge tube using a pipette, thereby extracting RNA. Meanwhile, the magnetic beads in the original centrifuge tube, which are adsorbed with cell tissue, are further supplemented with proteinase K and lysis buffer DTL. By controlling the temperature and time, DNA is released into the solution, thereby extracting DNA.

[0012] In this invention, the magnetic separation device is a device that uses a magnetic field to transfer or concentrate magnetic beads, specifically including magnetic frames or magnetic rods.

[0013] This invention involves dewaxing with xylene or a xylene substitute, followed by the addition of magnetic beads. This causes cell tissue to adhere to the surface of the magnetic beads. Under the influence of the magnetic field of the magnetic separation device, the magnetic beads carrying the cell tissue move directionally, achieving separation of the cell tissue from the solution. This allows for the lysis, binding, washing, and elution of the cell tissue, thus enabling high-throughput, automated extraction of nucleic acids.

[0014] Compared with the traditional magnetic bead method, the technical solution of this invention has the following advantages:

[0015] 1. Avoid loss of cells and tissues during nucleic acid extraction: Magnetic beads can confine cells and tissues within a certain space through a magnetic separation device, which can effectively prevent the tissues from being drawn away along with the dewaxing agent or other solutions;

[0016] 2. Facilitates automation: Magnetic beads can confine cells and tissues within a certain space through magnetic separation devices, effectively preventing cells and tissues from suspending in dewaxing agents or other solutions and clogging the pipetting devices in automated instruments, thus making automation more convenient.

[0017] 3. Easy to operate: During the dewaxing process, magnetic beads and magnetic separation devices can be used to separate cell tissues from dewaxing agents or other solutions without centrifugation or the use of a centrifuge, making it convenient to operate. Attached Figure Description

[0018] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0019] Figure 1 This is a comparison chart of DNA yield using column extraction and magnetic bead extraction. The black-filled column represents a commercially available column extraction method, while the white-filled column represents the automated method of this invention.

[0020] Figure 2 The graph shows a comparison of Ct values ​​between column extraction and magnetic bead-based fluorescent PCR. The black-filled column represents a commercially available column extraction method, while the white-filled column represents the automated method of this invention.

[0021] Figure 3 This graph compares RNA yield using column extraction and magnetic bead extraction. Black-filled columns represent a commercially available column extraction method, while white-filled columns represent the magnetic bead extraction method of this invention.

[0022] Figure 4 This is a comparison graph of Ct values ​​for fluorescent PCR using column extraction and magnetic bead methods. The black-filled column represents a commercially available column extraction method, while the white-filled column represents the magnetic bead method of this invention.

[0023] Figure 5 For the DNA and RNA yields of column extraction and magnetic bead extraction, the black-filled column represents a commercially available column extraction method, and the white-filled column represents the magnetic bead extraction method of this invention.

[0024] Figure 6 This is a comparison graph of Ct values ​​for DNA and RNA fluorescence PCR using column extraction and magnetic bead methods; the black-filled column represents a commercially available column extraction method, and the white-filled column represents the magnetic bead method of this invention.

[0025] Figure 7 This is a schematic diagram of the dewaxing process of the present invention. 1. Add a dewaxing agent to the FFPE sample; 2. Add magnetic beads to the solution containing the dewaxing agent; 3. Place a magnetic rack to remove the dewaxing agent and wax; 4. Volatilize the residual organic solvent. Detailed Implementation

[0026] Method 1: Extracting DNA from FFPE Samples

[0027] 1) Take 1 to 5 FFPE samples into a centrifuge tube and add 200 to 1000 μL of dewaxing agent (the types of dewaxing agents can be wide, the most common being xylene and xylene substitutes, such as alkanes, mineral oil, salami oil and citrus extracts, etc.);

[0028] 2) Mix well at room temperature or under heating for 5-10 minutes. The heating temperature range is 30-80℃, and 50-65℃ is better. Then dewax the FFPE sample.

[0029] 3) Add 20-100 μL of magnetic beads. A wide variety of magnetic beads can be used, including cellulose magnetic beads, hydroxyl magnetic beads, carboxyl magnetic beads, agarose magnetic beads, and silica-based glass magnetic beads. Among them, cellulose magnetic beads and hydroxyl-modified magnetic beads are more effective.

[0030] 4) After adding the magnetic beads, mix thoroughly at room temperature for 1–5 minutes to ensure full contact between the magnetic beads and the cells / tissues in the dewaxing agent. Place the centrifuge tube on a magnetic rack or insert a magnetic rod into the centrifuge tube. Let stand for 1–5 minutes until the magnetic beads have completely transferred to one side of the magnetic rack or rod, then remove all the dewaxing agent using a pipette.

[0031] 5) Add 500–1000 μL of organic solvent to the centrifuge tube. Commonly used organic solvents include anhydrous ethanol, acetone, and isopropanol. After adding the organic solvent, mix thoroughly at room temperature for 1–5 minutes. Place the centrifuge tube on a magnetic rack or insert a magnetic rod into the centrifuge tube. Let it stand for 1–5 minutes until the magnetic beads have completely transferred to one side of the magnetic rack or magnetic rod. Then, use a pipette to remove all the organic solvent.

[0032] 6) Heating at room temperature or for 5-10 minutes, with a heating temperature range of 30-80℃ and 50-65℃ being more effective, reduces the amount of residual organic solvent on the surface of magnetic beads and cell tissues.

[0033] 7) Add 10-50 μL of proteinase K and 100-400 μL of lysis buffer DTL (80-150 mM Tris·Cl, 40-70 mM EDTA, 80-150 mM dithiothreitol, 3-10% (v / v) sodium dodecyl sulfate, 10-25% (v / v) Tween 20), and place it at a certain heating temperature (the heating temperature range is 30-80℃, and 50-65℃ is better) to lyse the cell tissue and release the DNA into the solution;

[0034] 8) After digestion and lysis for 1-2 hours, wait for the solution temperature to drop to room temperature, add 100-400 μL of binding buffer DTB (80-150 mM Tris·Cl, 40-70 mM EDTA, 0.6-1.2 M KCl, 4-6 M guanidine isothiocyanate) and 50-80% ethanol, mix thoroughly for 1-5 minutes, and allow the DNA to adsorb onto the surface of the magnetic beads in the binding environment;

[0035] 9) Place the centrifuge tube on a magnetic rack or insert a magnetic rod into the centrifuge tube. Let it stand for 1–5 minutes until the magnetic beads have completely transferred to one side of the magnetic rack or magnetic rod, then use a pipette to remove all the solution from the tube;

[0036] 10) Add 500-1000 μL of washing buffer DW1 (150-400 mM Tris·Cl, 40-100 mM EDTA, 2-5 M guanidine isothiocyanate, 40-70% ethanol) to the centrifuge tube.

[0037] 11) Mix thoroughly at room temperature for 1–5 minutes, then place the centrifuge tube on a magnetic rack or insert a magnetic rod into the centrifuge tube. Let it stand for 1–5 minutes until the magnetic beads have completely transferred to one side of the magnetic rack or magnetic rod, then use a pipette to remove all the solution from the tube;

[0038] 12) Add 500-1000 μL of washing buffer DW2 (300-700 mM Tris·Cl, 0.8-1.2 M NaCl, 70-90% ethanol) to the centrifuge tube;

[0039] 13) Mix thoroughly at room temperature for 1–5 minutes. Place the centrifuge tube on a magnetic rack or insert a magnetic rod into the centrifuge tube. Let it stand for 1–5 minutes until the magnetic beads have completely transferred to one side of the magnetic rack or magnetic rod. Then, use a pipette to remove all the solution from the tube. This process is used to wash the magnetic beads and nucleic acids, reducing the residual amounts of proteins, lipids, and other organic matter and salts in the magnetic beads and nucleic acids, resulting in DNA with higher purity.

[0040] 14) Finally, add 30-200 μL of elution buffer DTE (10-20 mM Tris·Cl, 5-10 mM EDTA) to the centrifuge tube and mix thoroughly with the magnetic beads adsorbed with nucleic acids at room temperature for 1-5 min. The nucleic acids will gradually transfer from the surface of the magnetic beads to the solution.

[0041] 15) Place the centrifuge tube on a magnetic rack or insert a magnetic rod into the centrifuge tube. Let it stand for 1-5 minutes until the magnetic beads have completely transferred to one side of the magnetic rack or magnetic rod. Then, use a pipette to remove all the solution from the tube. The resulting solution is the DNA extracted from the FFPE sample.

[0042] Example 1: DNA Extraction from FFPE Samples

[0043] 1. Take two lung cancer FFPE samples into centrifuge tubes and add 1000 μL of xylene;

[0044] 2. Dewax by heating in a metal bath at 50℃ for 5 minutes;

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

[0046] 4. After adding the magnetic beads, mix thoroughly at room temperature for 1 minute to ensure full contact between the magnetic beads and the cells / tissues in the dewaxing agent. Place the centrifuge tube on a magnetic rack or insert a magnetic rod into the centrifuge tube. Let stand for 5 minutes to allow the magnetic beads to completely transfer to the magnetic rack, then remove all the dewaxing agent using a pipette.

[0047] 5. Add 1 mL of anhydrous ethanol to the centrifuge tube. After adding the organic solvent, mix thoroughly at room temperature for 1 min, then place the centrifuge tube on a magnetic rack. Let it stand for 5 min until the magnetic beads have completely transferred to the magnetic rack, then remove all the organic solvent using a pipette.

[0048] 6. After 5 minutes of ethanol evaporation at room temperature, the residual amount of organic solvent on the surface of magnetic beads and cell tissues is reduced;

[0049] 7. Add 20 μL of protease and 180 μL of lysis buffer DTL, heat in a 60°C metal bath for 2 hours to lyse the cell tissue and release DNA into the solution;

[0050] 8. After digestion and lysis for 2 hours, wait for the solution temperature to drop to room temperature, add the binding buffer DTB containing 50% organic solvent, mix thoroughly for 1 minute, and allow the DNA to adsorb onto the surface of the magnetic beads in the binding environment.

[0051] 9. Place the centrifuge tube on the magnetic rack. Let it stand for 5 minutes to allow the magnetic beads to completely transfer to the rack, then use a pipette to remove all the solution from the tube;

[0052] 10. Add 600 μL of washing buffer DW1 to the centrifuge tube;

[0053] 11. Mix thoroughly at room temperature for 1 minute, then place the centrifuge tube on a magnetic rack. Let stand for 5 minutes until the magnetic beads have completely transferred to the magnetic rack, then use a pipette to remove all the solution from the tube;

[0054] 12. Add 600 μL of washing buffer DW2 to the centrifuge tube;

[0055] 13. Mix thoroughly at room temperature for 1 minute, then place the centrifuge tube on a magnetic rack. Let it stand for 5 minutes until the magnetic beads have completely transferred to the rack. Use a pipette to remove all the solution from the tube. This process is used to wash the magnetic beads and nucleic acids, reducing the residual amounts of proteins, lipids, and other organic matter and salts in the beads and nucleic acids, resulting in DNA with higher purity.

[0056] 14. Finally, add 100 μL of elution buffer DTE to the centrifuge tube and mix thoroughly with the magnetic beads that adsorbed DNA at room temperature. The DNA will gradually transfer from the surface of the magnetic beads into the solution.

[0057] 15. Place the centrifuge tube on a magnetic rack or insert a magnetic bead into the centrifuge tube. Let it stand for 5 minutes until the magnetic beads have completely transferred to the magnetic rack. Then, use a pipette to remove all the solution from the tube. The resulting solution is the DNA extracted from the FFPE sample.

[0058] Figure 1-2 The graph shows a comparison of DNA yield and fluorescence PCR Ct value between column extraction and magnetic bead extraction. The black-filled column represents a commercially available column extraction method, while the white-filled column represents the automated method of this invention. The results show that the average DNA yield and fluorescence PCR Ct value obtained using this magnetic bead method are no less than those obtained using the column extraction method.

[0059] Method 2: Extracting RNA from FFPE samples

[0060] 1) Take 1 to 5 FFPE samples into a centrifuge tube and add 200 to 1000 μL of dewaxing agent (the types of dewaxing agents can be wide, the most common being xylene and xylene substitutes, alkanes, mineral oil, salami oil and citrus extract, etc.);

[0061] 2) Mix well at room temperature or under heating for 5-10 minutes. The heating temperature range is 30-80℃, and 50-65℃ is better. Then dewax the FFPE sample.

[0062] 3) Add 20-100 μL of magnetic beads. A wide variety of magnetic beads can be used, including cellulose magnetic beads, hydroxyl magnetic beads, carboxyl magnetic beads, agarose magnetic beads, and silica-based glass magnetic beads. Among them, cellulose magnetic beads and hydroxyl-modified magnetic beads are more effective.

[0063] 4) After adding the magnetic beads, mix thoroughly at room temperature for 1–5 minutes to ensure full contact between the magnetic beads and the cells / tissues in the dewaxing agent. Place the centrifuge tube on a magnetic rack or insert a magnetic rod into the centrifuge tube;

[0064] 5) Let it stand for another 1 to 5 minutes until the magnetic beads have completely transferred to one side of the magnetic rack or magnetic rod, then use a pipette to remove all the dewaxing agent.

[0065] 6) Add 500–1000 μL of organic solvent to the centrifuge tube. Commonly used organic solvents include anhydrous ethanol, acetone, and isopropanol. This gradually transitions the cell tissue from the organic solvent environment to the aqueous environment, restoring bound water in the cells. This facilitates the function of enzymes and compounds involved in digestion and lysis during subsequent nucleic acid extraction, improving the digestion and lysis efficiency and RNA release.

[0066] 7) After adding the organic solvent, mix thoroughly at room temperature for 1–5 minutes. Place the centrifuge tube on a magnetic rack or insert a magnetic rod into the centrifuge tube. Let it stand for 1–5 minutes until the magnetic beads have completely transferred to one side of the magnetic rack or magnetic rod. Then, use a pipette to remove all the organic solvent.

[0067] 8) Heat at room temperature or for 5-10 minutes. The heating temperature range is 30-80℃, and 50-65℃ is even better. This will fully volatilize the dewaxing agent and reduce the amount of residual organic solvent on the surface of the magnetic beads and cell tissues.

[0068] 9) Add 10–50 μL of proteinase K and 100–400 μL of lysis buffer RTL (containing 150–300 mM Tris·Cl, 0.8–1.2 M NaCl, 8–14% (V / V) Tween 20, and 0.2–0.8% (V / V) sodium dodecyl sulfate to a centrifuge tube. Place the tube in a specific heating temperature range (30–80 °C, with 50–65 °C being more effective) to lyse the cell tissue and release RNA into the solution.

[0069] 10) After digestion and lysis for 15-30 minutes, wait for the solution temperature to drop to room temperature, place the centrifuge tube on a magnetic rack or put a magnetic rod in the centrifuge tube, let it stand for 1-5 minutes until the magnetic beads are completely transferred to one side of the magnetic rack or magnetic rod, and then use a pipette to transfer the digestion and lysis solution containing RNA to a new centrifuge tube.

[0070] 11) Add 100–400 μL of binding buffer RTB (50–150 mM Tris·Cl, 150–250 mM EDTA, 0.8–1.2 M KCl, 0.4–1.2 M NaCl, 0.1–0.5% (v / v) dodecyl sulfate, 8–16% (v / v) Tween 20, 4–6 M guanidine isothiocyanate), 20–100 μL of magnetic beads, and 30–80% ethanol to the RNA-containing digestion and lysis buffer. Mix thoroughly to allow the RNA to adsorb onto the surface of the magnetic beads in the binding environment. Place the centrifuge tube on a magnetic rack or insert a magnetic rod into the centrifuge tube. Let it stand for 1–5 minutes until the magnetic beads have completely transferred to one side of the magnetic rack or magnetic rod. Then, use a pipette to remove all the solution from the tube.

[0071] 12) Add 500–1000 μL of washing buffer RTW (250–350 mM Tris·Cl, 30–70 mM EDTA, 0.5–1.5 M NaCl, 70–90% ethanol) to the centrifuge tube. This is used to wash the magnetic beads and nucleic acids, reducing the residual amounts of proteins, lipids, and other organic matter and salts in the magnetic beads and nucleic acids, thus obtaining RNA with higher purity.

[0072] 13) Mix thoroughly at room temperature for 1–5 minutes. Place the centrifuge tube on a magnetic rack or insert a magnetic rod into the centrifuge tube. Let stand for 1–5 minutes until the magnetic beads have completely transferred to one side of the magnetic rack or magnetic rod. Then, use a pipette to remove all the solution from the tube. Repeat the washing process 1–3 times.

[0073] 14) Finally, add 30-200 μL of elution buffer RTE (10-20 mM Tris·Cl, 5-20 mM EDTA) to the centrifuge tube and mix thoroughly with the magnetic beads adsorbed with nucleic acids at room temperature for 1-5 min. The nucleic acids will gradually transfer from the surface of the magnetic beads to the solution.

[0074] 15) Place the centrifuge tube on a magnetic rack or insert a magnetic rod into the centrifuge tube. Let it stand for 1–5 minutes until the magnetic beads have completely transferred to one side of the magnetic rack or magnetic rod. Then, use a pipette to remove all the solution from the tube. The resulting solution is the RNA extracted from the FFPE sample.

[0075] Example 2: RNA extraction from FFPE samples

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

[0077] 2. Dewax by heating in a metal bath at 50℃ for 5 minutes;

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

[0079] 4. After adding the magnetic beads, mix thoroughly for 5 minutes to ensure that the magnetic beads are in full contact with the cells and tissues in the dewaxing agent;

[0080] 5. Place the centrifuge tubes on the magnetic rack. Let them stand for 5 minutes to allow the magnetic beads to transfer completely to the rack, then use a pipette to remove all the dewaxing agent.

[0081] 6. Add 1 mL of anhydrous ethanol to the centrifuge tube. After adding the organic solvent, mix thoroughly for 1 min at room temperature. Place the centrifuge tube on a magnetic rack or insert a magnetic bar into the centrifuge tube. Let it stand for 5 min until the magnetic beads have completely transferred to the magnetic rack. Then, use a pipette to remove all the organic solvent.

[0082] 7. After 5 minutes of ethanol evaporation at room temperature, the residual amount of organic solvent on the surface of magnetic beads and cell tissues is reduced;

[0083] 8. Add 20 μL proteinase K and 160 μL lysis buffer RTL, heat in a 60℃ metal bath for 15 min to lyse the cell tissue and release RNA into the solution;

[0084] 9. After digestion and lysis for 15 minutes, wait for the solution temperature to drop to room temperature, place the centrifuge tube on a magnetic rack, and let it stand for 5 minutes until the magnetic beads are completely transferred to the magnetic rack. Use a pipette to transfer the digestion and lysis buffer containing RNA to a new centrifuge tube.

[0085] 10. Add 150 μL L / TbB, 850 μL anhydrous ethanol, and 20 μL magnetic beads to the RNA-containing digestion and lysis buffer. Mix thoroughly to allow the RNA to adsorb onto the surface of the magnetic beads in the binding environment. Place the centrifuge tube on a magnetic rack or insert a magnetic rod into the centrifuge tube. Let it stand for 5 minutes to allow the magnetic beads to completely transfer to the magnetic rack. Then, use a pipette to remove all the solution from the tube.

[0086] 11. Add 600 μL of RTW washing buffer to the centrifuge tube to wash the magnetic beads and nucleic acids. This reduces the residual amount of organic matter such as proteins and lipids and salts in the magnetic beads and nucleic acids, resulting in RNA with higher purity.

[0087] 12. Mix thoroughly at room temperature, then place the centrifuge tube on a magnetic rack. Let stand for 5 minutes to allow the magnetic beads to completely transfer to the rack. Use a pipette to remove all the solution from the tube. Repeat the washing process 1–3 times.

[0088] 13. Finally, add 100 μL of elution buffer RTE to the centrifuge tube and mix thoroughly with the magnetic beads adsorbed with nucleic acids at room temperature. The nucleic acids will gradually transfer from the surface of the magnetic beads into the solution.

[0089] 14. Place the centrifuge tube on a magnetic rack or insert a magnetic bead into the centrifuge tube. Let it stand for 5 minutes until the magnetic beads have completely transferred to the magnetic rack. Then, use a pipette to remove all the solution from the tube. The resulting solution is the RNA extracted from the FFPE sample.

[0090] Figure 3-4 The graph shows a comparison of RNA yield and fluorescence PCR Ct value between column extraction and magnetic bead extraction. The black-filled column represents a commercially available column extraction method, while the white-filled column represents the magnetic bead method of this invention. The results show that the average RNA yield and fluorescence PCR Ct value extracted using this magnetic bead method are no less than those of the column extraction method.

[0091] Method 3: Simultaneous extraction of DNA and RNA from FFPE samples

[0092] After dewaxing FFPE samples and rehydrating tissue cells, add 10–50 μL of proteinase K and 100–400 μL of lysis buffer RTL to centrifuge tubes. Briefly heat at a controlled temperature (30–80°C, with 50–65°C being more effective). By controlling the heating temperature and time, cell membranes rupture while the nuclear membrane and cytoskeleton remain intact. Since RNA is mainly found in the extracellular matrix, while DNA is mainly found in the nucleus, under these conditions, RNA is released into the lysis buffer due to cell membrane rupture. Because the nuclear membrane remains intact, DNA remains in the cytoskeleton, attached to the surface of magnetic beads along with the cell tissue. Place the centrifuge tube on a magnetic rack or insert a magnetic rod, and let it stand for 1–5 minutes until the magnetic beads are completely transferred to one side of the rack or rod. Then, use a pipette to transfer the lysis buffer containing RNA to a new centrifuge tube. Extract RNA using the nucleic acid binding, washing, and elution steps mentioned in Application 2. Meanwhile, 10–50 μL of proteinase K and 100–400 μL of lysis buffer DTL are added to the magnetic beads containing cells in the centrifuge tube. The tube is then heated at a specific temperature (30–80 °C, with 50–65 °C being more effective) to completely lyse the cells and release DNA into the solution. DNA extraction is then completed using the nucleic acid binding, washing, and elution steps mentioned in Application 1.

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

[0094] After dewaxing and rehydration of cells in Application 2, add 25 μL of proteinase K and 200 μL of lysis buffer RTL to centrifuge tubes and heat in a 60°C metal bath for 15 min. Since RNA is mainly found in the intercellular matrix, while DNA is mainly found in the cell nucleus, place the centrifuge tubes on a magnetic rack and let them stand for 5 min to allow the magnetic beads to completely transfer to the rack. Use a pipette to transfer 180 μL of RNA digestion lysis buffer to a new centrifuge tube. Use the nucleic acid binding, washing, and elution steps mentioned in Application 2 to complete RNA extraction. For the magnetic beads still adsorbed with cells and tissue, add another 15 μL of proteinase K and 140 μL of lysis buffer DTL, and heat in a 60°C metal bath for 1–2 h to completely lyse the cells and tissue, releasing DNA into the solution. Use the nucleic acid binding, washing, and elution steps mentioned in Application 1 to complete DNA extraction.

[0095] Figure 5-6 The graph shows a comparison of DNA and RNA yields and fluorescence PCR Ct values ​​between column extraction and magnetic bead extraction. The black-filled column represents a commercially available column extraction method, while the white-filled column represents the magnetic bead method of this invention. The results show that the average DNA and RNA yields and fluorescence PCR Ct values ​​extracted using this magnetic bead method are no less than those of the column extraction method.

Claims

1. A method for extracting nucleic acids from FFPE samples, wherein the extraction of nucleic acids from FFPE samples involves extracting DNA or RNA separately from FFPE. First, a dewaxing agent is added to the FFPE sample. After dewaxing, magnetic beads are added to ensure full contact between the magnetic beads and the cell tissue in the dewaxing agent, allowing the cell tissue to adhere to the surface of the magnetic beads. Under the action of the magnetic field of a magnetic separation device, the magnetic beads adsorbing the cell tissue will directionally move the cell tissue carrying its surface to the magnetic separation device. Then, the dewaxing agent is removed, achieving separation of the cell tissue from the solution and preventing the tissue from being absorbed along with the dewaxing agent or other solutions, thereby extracting DNA or RNA from the FFPE. The magnetic beads are cellulose magnetic beads or hydroxyl-modified magnetic beads.

2. The method for extracting nucleic acids from FFPE samples as described in claim 1, comprising the following steps: 1) Take 1-5 FFPE samples into a centrifuge tube and add 200-1000 μL of dewaxing agent; 2) Mix at room temperature or under heating for 5-10 minutes, with the heating temperature range being 30-80℃, to dewax the FFPE sample; 3) Add 20~100 μL of magnetic beads; 4) After adding the magnetic beads, mix thoroughly at room temperature for 1-5 minutes to ensure that the magnetic beads are in full contact with the cells and tissues in the dewaxing agent; place the centrifuge tube on a magnetic rack or put a magnetic rod in the centrifuge tube; let stand for 1-5 minutes until the magnetic beads are completely transferred to one side of the magnetic rack or magnetic rod, and then remove all the dewaxing agent with a pipette. 5) Add 500-1000 μL of organic solvent to the centrifuge tube; after adding the organic solvent, mix thoroughly at room temperature for 1-5 minutes, place the centrifuge tube on a magnetic rack or put a magnetic rod in the centrifuge tube; let stand for 1-5 minutes, and after the magnetic beads have completely transferred to one side of the magnetic rack or magnetic rod, remove all the organic solvent with a pipette. 6) Reduce the amount of residual organic solvent on the surface of magnetic beads and cell tissue by heating at room temperature or heating for 5-10 minutes at a temperature range of 30-80°C. 7) Add 10-50 μL of proteinase K and 100-400 μL of lysis buffer DTL, or add 10-50 μL of proteinase K and 100-400 μL of lysis buffer RTL; 8) Extract DNA or RNA.

3. A method for extracting nucleic acids from FFPE samples, wherein the extraction of nucleic acids from FFPE samples involves simultaneously extracting DNA and RNA from FFPE. First, a dewaxing agent is added to the FFPE sample. After dewaxing, magnetic beads, proteinase K, and lysis buffer RTL are added to a centrifuge tube. By controlling the temperature and time, the cell membrane ruptures while the nuclear membrane, cytoskeleton, and other structures remain intact, allowing RNA to be released into the solution. The centrifuge tube is then placed on a magnetic separation device, and the digestion lysis buffer containing RNA is transferred to a new centrifuge tube using a pipette for RNA extraction. Meanwhile, the magnetic beads containing cell tissue in the original centrifuge tube are further supplemented with proteinase K and lysis buffer DTL. By controlling the temperature and time, DNA is released into the solution for DNA extraction. The method includes the following steps: 1) Take 1-5 FFPE samples into a centrifuge tube and add 200-1000 μL of dewaxing agent; 2) Mix at room temperature or under heating for 5-10 minutes, with the heating temperature range being 30-80℃, to dewax the FFPE sample; 3) Add 20~100 μL of magnetic beads; the magnetic beads are cellulose magnetic beads or hydroxyl-modified magnetic beads; 4) After adding the magnetic beads, mix thoroughly at room temperature for 1-5 minutes to ensure that the magnetic beads are in full contact with the cells and tissues in the dewaxing agent; place the centrifuge tube on a magnetic rack or put a magnetic rod in the centrifuge tube; let stand for 1-5 minutes until the magnetic beads are completely transferred to one side of the magnetic rack or magnetic rod, and then remove all the dewaxing agent with a pipette. 5) Add 500-1000 μL of organic solvent to the centrifuge tube; after adding the organic solvent, mix thoroughly at room temperature for 1-5 minutes, place the centrifuge tube on a magnetic rack or put a magnetic rod in the centrifuge tube; let stand for 1-5 minutes, and after the magnetic beads have completely transferred to one side of the magnetic rack or magnetic rod, remove all the organic solvent with a pipette. 6) Reduce the amount of residual organic solvent on the surface of magnetic beads and cell tissue by heating at room temperature or heating for 5-10 minutes at a temperature range of 30-80°C. 7) Add 10-50 μL of proteinase K and 100-400 μL of lysis buffer DTL, or add 10-50 μL of proteinase K and 100-400 μL of lysis buffer RTL; 8) Extract DNA and RNA.

4. The method for extracting nucleic acids from FFPE samples as described in claim 2, characterized in that: Step 8) DNA extraction is performed, which includes: 9) Add 10-50 μL of proteinase K and 100-400 μL of lysis buffer DTL to a centrifuge tube; lyse the cell tissue at a heating temperature of 50-65°C to release DNA into the solution; the lysis buffer DTL is composed of the following components: 80-150 mM Tris•Cl, 40-70 mM EDTA, 80-150 mM dithiothreitol, 3-10% (v / v) sodium dodecyl sulfate and 10-25% (v / v) Tween 20; 10) Lyse the cell tissue at a heating temperature of 50~65℃ to release the DNA into the solution; 11) After digestion and lysis for 1-2 h, wait for the solution temperature to drop to room temperature, add 100-400 μL of binding buffer DTB and 50-80% ethanol, mix thoroughly, and allow the DNA to adsorb onto the surface of the magnetic beads in the binding environment; the binding buffer DTB is composed of the following components: 80-150 mM Tris·Cl, 40-70 mM EDTA, 0.6-1.2 M KCl and 4-6 M guanidine isothiocyanate; 12) After standing at room temperature for 1-5 minutes, place the centrifuge tube on a magnetic rack or put a magnetic rod in the centrifuge tube; after standing for 1-5 minutes until the magnetic beads have completely transferred to one side of the magnetic rack or magnetic rod, use a pipette to remove all the solution in the tube. 13) Add 500-1000 μL of washing buffer DW1 to the centrifuge tube. The washing buffer DW1 is composed of the following components: 150-400 mM Tris·Cl, 40-100 mM EDTA, 2-5 M guanidine isothiocyanate and 40-70% ethanol. 14) Mix thoroughly at room temperature for 1-5 minutes, place the centrifuge tube on a magnetic rack or put a magnetic rod in the centrifuge tube; let stand for 1-5 minutes until the magnetic beads are completely transferred to one side of the magnetic rack or magnetic rod, then use a pipette to remove all the solution in the tube. 15) Add 500-1000 μL of washing buffer DW2 to the centrifuge tube. The washing buffer DW2 is composed of the following components: 300-700 mM Tris·Cl, 0.8-1.2 M NaCl and 70-90% ethanol. 16) Mix thoroughly at room temperature for 1-5 minutes, place the centrifuge tube on a magnetic rack or put a magnetic rod in the centrifuge tube; let stand for 1-5 minutes until the magnetic beads are completely transferred to one side of the magnetic rack or magnetic rod, then use a pipette to remove all the solution in the tube. 17) Finally, add 30-200 μL of elution buffer DTE to the centrifuge tube and mix thoroughly with the magnetic beads adsorbed with nucleic acids at room temperature for 1-5 min. The nucleic acids will gradually transfer from the surface of the magnetic beads to the solution. The buffer DTE is composed of the following components: 10-20 mM Tris·Cl and 5-10 mM EDTA. 18) Place the centrifuge tube on a magnetic rack or put a magnetic rod into the centrifuge tube; let it stand for 1-5 minutes until the magnetic beads are completely transferred to one side of the magnetic rack or magnetic rod, then use a pipette to remove all the solution in the tube. The resulting solution is the DNA extracted from the FFPE sample.

5. The method for extracting nucleic acids from FFPE samples as described in claim 2, characterized in that: Step 8) Perform RNA extraction, including the following steps: 9) Add 10-50 μL of proteinase K and 100-400 μL of lysis buffer RTL to a centrifuge tube, and lyse the cell tissue at a heating temperature of 50-65°C to release RNA into the solution; the buffer RTL is composed of the following components: 150-300 mM Tris·Cl, 0.8-1.2 M NaCl, 8-14% (V / V) Tween 20 and 0.2-0.8% (V / V) sodium dodecyl sulfate; 10) After digestion and lysis for 15-30 min, wait for the solution temperature to drop to room temperature, place the centrifuge tube on a magnetic rack or put a magnetic rod in the centrifuge tube, let it stand for 1-5 min until the magnetic beads are completely transferred to one side of the magnetic rack or magnetic rod, and then use a pipette to transfer the digestion and lysis solution containing RNA to a new centrifuge tube. 11) Add 100-400 μL of binding buffer RTB, 20-100 μL of magnetic beads, and 30-80% ethanol to the RNA-containing digestion and lysis buffer. Mix thoroughly to allow the RNA to adsorb onto the surface of the magnetic beads in the binding environment. Place the centrifuge tube on a magnetic rack or insert a magnetic rod into the centrifuge tube. Let it stand for 1-5 minutes until the magnetic beads are completely transferred to one side of the magnetic rack or magnetic rod. Then, use a pipette to remove all the solution from the tube. The binding buffer RTB is composed of the following components: 50-150 mM Tris·Cl, 150-250 mM EDTA, 0.8-1.2 M KCl, 0.4-1.2 M NaCl, 0.1-0.5% (V / V) dodecyl sulfate, 8-16% (V / V) Tween 20, and 4-6 M guanidine isothiocyanate. 12) Add 500-1000 μL of washing buffer RTW to the centrifuge tube. The washing buffer RTW is composed of the following components: 250-350 mM Tris·Cl, 30-70 mM EDTA, 0.5-1.5 M NaCl and 70-90% ethanol. 13) Mix thoroughly at room temperature for 1-5 minutes, place the centrifuge tube on a magnetic rack or put a magnetic rod in the centrifuge tube; let stand for 1-5 minutes until the magnetic beads are completely transferred to one side of the magnetic rack or magnetic rod, then use a pipette to remove all the solution in the tube; repeat the washing process 1-3 times. 14) Finally, add 30-200 μL of elution buffer RTE to the centrifuge tube and mix thoroughly with the magnetic beads adsorbed with nucleic acids at room temperature for 1-5 min. The nucleic acids will gradually transfer from the surface of the magnetic beads to the solution. The elution buffer RTE is composed of the following components: 10-20 mM Tris·Cl and 5-20 mM EDTA. 15) Place the centrifuge tube on a magnetic rack or put a magnetic rod into the centrifuge tube; let it stand for 1-5 minutes until the magnetic beads are completely transferred to one side of the magnetic rack or magnetic rod, then use a pipette to remove all the solution in the tube. The resulting solution is the RNA extracted from the FFPE sample.

6. The method for extracting nucleic acids from FFPE samples according to claim 3, characterized in that: Step 8) Simultaneous extraction of DNA and RNA, including the following steps: After dewaxing FFPE samples and rehydrating tissue cells, 10-50 μL of proteinase K and 100-400 μL of lysis buffer RTL were added to centrifuge tubes and briefly heated at 50-65°C. By controlling the heating temperature and heating time, the cell membrane was ruptured while the nuclear membrane, cytoskeleton and other structures remained intact. Place the centrifuge tube on a magnetic rack or insert a magnetic rod into the centrifuge tube. Let it stand for 1-5 minutes until the magnetic beads are completely transferred to one side of the magnetic rack or magnetic rod. Then, use a pipette to transfer the digestion lysis buffer containing RNA into a new centrifuge tube. Use steps 9)-18) of claim 5 to complete the RNA extraction. For the magnetic beads containing cell tissue adsorbed in the centrifuge tube, add 10-50 μL of proteinase K and 100-400 μL of lysis buffer DTL. Place the tube at a heating temperature of 50-65°C to completely lyse the cell tissue and release DNA into the solution. Use steps 10-15) of claim 4 to complete the DNA extraction.

7. The method for extracting nucleic acids from FFPE samples according to claim 2 or 3, characterized in that: The heating temperature for steps 2) and / or 6) is 50~65℃.

8. The method for extracting nucleic acids from FFPE samples according to claim 2 or 3, characterized in that: The organic solvent mentioned in step 5) includes at least one of anhydrous ethanol, acetone, and isopropanol.

9. The method for extracting nucleic acids from FFPE samples according to claim 2 or 3, characterized in that: Step 7) The lysis buffer RTL is composed of the following components: 150-300 mM Tris•Cl, 0.8-1.2 M NaCl, 8-14% (V / V) Tween 20 and 0.2-0.8% (V / V) sodium dodecyl sulfate.

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