Method for extracting DNA / RNA (deoxyribonucleic acid / ribonucleic acid) from formalin-fixed paraffin-embedded tissue

Abstract

The invention discloses a method for extracting DNA / RNA (deoxyribonucleic acid / ribonucleic acid) from formalin-fixed paraffin-embedded tissue, and relates to the technical field of biotechnology and molecular diagnose.The method includes dissolving low-polarity fatty acid ester in a dewaxing agent with paraffin, quickly dissolving a paraffin wrapping layer under mild heating conditions, and extracting DNA / RNA from the paraffin wrapping layer after dewaxing is completed. The method does not need multiple times of centrifugation, absorption discarding and ethanol washing to physically remove an organic solvent, a subsequent aqueous lysis solution is directly added, and the dewaxing agent is low in density and stably suspends on an aqueous phase to form a liquid covering layer, so that in the whole subsequent cracking and high-temperature incubation process, evaporation of an aqueous-phase reagent is effectively prevented, and the stability of the dewaxing agent is improved. Compared with the prior art, the method has the advantages that the reaction system stability is guaranteed, physical loss of trace tissue fragments or released nucleic acid caused by repeated transfer and liquid cleaning is thoroughly avoided, meanwhile, the dewaxing agent is non-toxic or low in toxicity, high in boiling point and low in volatility, and health risks of operators and environmental hazards caused by using xylene are fundamentally eliminated.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and molecular diagnostics, specifically a method for extracting DNA / RNA from formalin-fixed paraffin-embedded tissues. Background Technology

[0002] Formalin-fixed paraffin embedding (FFPE) is the standard method for preserving tissue samples in clinical pathology diagnosis, and it is widely used in disease mechanism research, biomarker screening, and molecular diagnostics. With the development of precision medicine, molecular detection technologies based on FFPE samples, such as PCR, qPCR, Sanger sequencing, and high-throughput sequencing, are increasingly used in personalized cancer treatment and genetic disease diagnosis. Therefore, obtaining high-purity, high-integrity nucleic acids (DNA / RNA) from FFPE samples is a prerequisite for ensuring the accuracy of downstream detection.

[0003] However, nucleic acid extraction from FFPE samples faces significant technical challenges. First, traditional dewaxing methods typically use xylene, which is highly toxic, volatile, and carcinogenic. Furthermore, the process involves multiple centrifugations and ethanol washings, a cumbersome procedure that easily leads to the physical loss of trace amounts of sample. Second, formalin fixation causes methylene cross-links between nucleic acids and proteins, hindering polymerase amplification. High-temperature heating is necessary to reverse these cross-links. However, under high-temperature conditions, trace amounts of divalent metal ions remaining in the sample can catalyze the breakage of phosphodiester bonds, leading to severe DNA / RNA degradation and making it difficult to meet the requirements for long-fragment sequencing.

[0004] Existing technologies have attempted to replace xylene with mineral oil and limonene, which reduces toxicity but suffers from problems such as high viscosity, difficulty in absorption, and unpleasant odor. Furthermore, the activity of proteinase K depends on the formulation design of the lysis buffer. To maintain its structural stability, high concentrations of metal chelating agents can inhibit its activity, leading to incomplete protein digestion; if the chelating agent is reduced, nucleic acids cannot be protected from hydrolysis catalyzed by metal ions in subsequent high-temperature steps.

[0005] Therefore, there is an urgent need in this field for an innovative extraction solution that can achieve safe and efficient dewaxing while balancing the needs for enzyme activity and high-temperature protection of nucleic acids. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for extracting DNA / RNA from formalin-fixed paraffin-embedded tissues. This method utilizes the miscibility of low-polarity fatty acid esters in the dewaxing agent with paraffin, rapidly dissolving the paraffin coating under gentle heating conditions. After dewaxing, there is no need for repeated centrifugation, aspiration, and ethanol washing to physically remove organic solvents; the subsequent aqueous lysis buffer is directly added. Due to the low density of the dewaxing agent, it stably suspends above the aqueous phase, forming a liquid coating layer. Throughout the subsequent lysis and high-temperature incubation process, this not only effectively prevents the evaporation of aqueous reagents and ensures the stability of the reaction system, but also completely avoids the physical loss of trace tissue fragments or released nucleic acids caused by repeated transfer and washing of liquids. Furthermore, the dewaxing agent itself is non-toxic or low-toxic, with a high boiling point and low volatility, fundamentally eliminating the health risks to operators and environmental hazards associated with the use of xylene.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for extracting DNA / RNA from formalin-fixed paraffin-embedded tissues, the steps of which are as follows: S100. Formalin-fixed paraffin-embedded tissue section samples are mixed with a dewaxing agent and heated and incubated to fully dissolve the paraffin. The dewaxing agent contains fatty acid esters with the general formula R1-COO-R2, wherein R1 is an aliphatic hydrocarbon group of C11-C18, and the aliphatic hydrocarbon group is saturated or unsaturated, straight-chain or branched, and R2 is a hydrocarbon group of C1-C8. S200: Add tissue lysis buffer and proteinase K to S100, and perform tissue lysis at 30-70°C. The tissue lysis buffer contains buffer compounds and anionic surfactants, but does not contain metal ion chelating agents. The concentration of proteinase K is 20 mg / mL. S300. After tissue lysis is completed, a reverse cross-linking protective solution is added and mixed evenly to chelate metal ions to obtain a mixture. The reverse cross-linking protective solution contains a metal ion chelating agent. S400: The mixture obtained in S300 is incubated at 80℃-100℃ to reverse the methylene crosslinking between nucleic acids and proteins; S500. After heat incubation, the mixture is centrifuged to separate into layers. The lower aqueous phase containing DNA / RNA is retained and the upper organic phase is discarded. Then, a binding solution is added to the retained lower aqueous phase to adsorb DNA / RNA onto the solid adsorption carrier. After washing with washing solution I and washing solution II in sequence, the DNA / RNA is eluted with elution solution to obtain purified DNA / RNA.

[0008] Further, in S100, the fatty acid ester of the dewaxing agent is selected from isopropyl myristate, 2-ethylhexyl palmitate, isopropyl palmitate, ethyl oleate, or a mixture thereof.

[0009] Furthermore, in S100, the dewaxing agent also contains an oil-soluble indicator dye, which is selected from dyes whose chromophores have anthraquinone or azo structures, including Solvent Blue 35, Solvent Green 3, Sudan Black B, and Disperse Blue 14, to clearly indicate the oil-water interface and prevent accidental aspiration during operation.

[0010] Furthermore, in S200, the buffer compound of the tissue lysis buffer is selected from one or more of N-(tris(hydroxymethyl)methyl)glycine, tris(hydroxymethyl)aminomethane, di(2-hydroxyethyl)iminotris(hydroxymethyl)methane, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), N-cyclohexyl-2-aminoethanesulfonic acid, N,N-bis(2-hydroxyethyl)glycine, 2-(N-morpholino)ethanesulfonic acid, piperazine-1,4-bis(2-ethanesulfonic acid), 3-(N-morpholino)propanesulfonic acid, and phosphate buffer. The concentration of the buffer compound is ≤500mM, which is used to maintain the pH stability of the lysis system and provide a suitable reaction environment for proteinase K.

[0011] Furthermore, in S200, the anionic surfactant of the tissue lysis solution is selected from one or more of dodecyl sarcosine, sodium N-lauroyl sarcosine, sodium dodecyl sulfate, sodium dodecyl sulfonate, and sodium dodecylbenzene sulfonate. The concentration of the anionic surfactant is 0.1%-10%, and it is used to disrupt the structure of tissue cell membranes and nuclear membranes and promote the release of nucleic acids.

[0012] Furthermore, in S300, the reverse crosslinking protective liquid contains a metal ion chelating agent; The metal ion chelating agent is selected from one or more of ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid-ethylene glycol, citrate, diethylenetriaminepentaacetic acid, or aminotriacetic acid; After adding the reverse crosslinking protective solution, the final concentration of the metal ion chelating agent in the reaction system is 0.1mM-50mM, which is used to chelate divalent metal ions in the system and avoid the catalytic hydrolysis of nucleic acids by metal ions at high temperatures.

[0013] Furthermore, in S300, the reverse crosslinking protective liquid also contains monovalent salt ions; The monovalent salt ion is selected from one or more of lithium chloride, sodium chloride, or potassium chloride; After adding the reverse crosslinking protection solution, the final concentration of monovalent salt ions in the reaction system is 1mM-100mM, which is used to maintain the stability of the ionic strength of the system and help protect the nucleic acid structure.

[0014] Furthermore, in S500, the binding solution is a buffer solution containing a high concentration of ionizing salt, nonionic surfactant, and organic solvent; The washing solution I is a buffer solution containing liquid salt, buffer and alcohol; The washing solution II is a buffer solution containing buffer and alcohol; The elution buffer is a low-salt buffer solution; The solid-phase adsorption carrier is an adsorption column or magnetic beads.

[0015] Furthermore, in the processes from S100 to S400, there is no need to physically remove the dewaxing agent. Because the dewaxing agent has a density less than water, it remains stable on the upper layer of the aqueous phase after the addition of tissue lysis solution and reverse crosslinking protection solution, forming a liquid covering layer. The liquid covering layer can prevent the aqueous phase from evaporating during subsequent thermal incubation, ensuring the stability of the reaction system concentration.

[0016] Compared with existing technologies, this method for extracting DNA / RNA from formalin-fixed paraffin-embedded tissues has the following advantages: I. This invention utilizes the low-polarity fatty acid esters of the dewaxing agent to dissolve paraffin wax rapidly under mild heating conditions. After dewaxing, there is no need for multiple centrifugations, aspiration, and ethanol washing to physically remove organic solvents. The dewaxing agent can be directly added to the subsequent aqueous lysis buffer. Due to its low density, the dewaxing agent is stably suspended on the aqueous phase, forming a liquid coating layer. Throughout the subsequent lysis and high-temperature incubation process, this not only effectively prevents the evaporation of aqueous reagents and ensures the stability of the reaction system, but also completely avoids the physical loss of trace tissue fragments or released nucleic acids caused by repeated transfer and washing of liquids. At the same time, the dewaxing agent itself is non-toxic or low-toxic, with a high boiling point and low volatility, fundamentally eliminating the health risks to operators and environmental hazards associated with the use of xylene.

[0017] II. This invention employs a segmented ion protection strategy. During the tissue lysis stage, a tissue lysis buffer without metal ion chelating agents is used to provide proteinase K-dependent lysis. The highly active environment enables it to efficiently digest histones that are cross-linked with nucleic acids to release nucleic acids. Before high-temperature reversal cross-linking, a reversal cross-linking protection solution containing a high concentration of metal ion chelating agent is added to rapidly chelate divalent metal ions in the system, eliminating the catalytic conditions for nucleic acid hydrolysis at high temperatures. At the same time, monovalent salt ions maintain the stability of the ionic strength of the system, which not only solves the contradiction between proteinase K activity and nucleic acid protection, but also effectively preserves the integrity of nucleic acids, meeting the detection requirements of downstream long-fragment sequencing.

[0018] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0020] Figure 1 A flowchart illustrating the steps of a method for extracting DNA / RNA from formalin-fixed paraffin-embedded tissues; Figure 2 This is a comparison diagram of the interfacial layering between the dewaxing agent and the aqueous system in an embodiment of the present invention; Figure 3 These are agarose gel electrophoresis images of nucleic acids extracted under different ion protection strategies in embodiments of the present invention. Detailed Implementation

[0021] To better understand the above technical solutions, a detailed description of the solutions will be provided below in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0022] To address the problems in existing formalin-fixed paraffin-embedded tissue nucleic acid extraction processes, such as the high toxicity of dewaxing agents, cumbersome procedures leading to trace sample loss, and the conflicting effects of high-temperature reversal of cross-linking and proteinase K activity, as well as the easy degradation of nucleic acids, this invention first describes its application scenarios. This invention is primarily applied in clinical pathology diagnosis, personalized cancer treatment, genetic disease testing, and biomarker screening, specifically targeting the DNA / RNA extraction needs of formalin-fixed paraffin-embedded tissue samples, particularly for applications requiring nucleic acid purity ≥95% and integrity sufficient for downstream detection such as PCR and high-throughput sequencing. In these scenarios, the xylene dewaxing agent used in traditional extraction techniques is highly toxic, volatile, and carcinogenic, and the multiple centrifugation and washing steps easily cause physical loss of trace samples, such as those obtained through puncture biopsies. Furthermore, the methylene cross-linking formed by formalin fixation requires high-temperature reversal, but residual divalent metal ions at high temperatures catalyze nucleic acid hydrolysis, while the addition of metal chelating agents inhibits proteinase K activity, leading to incomplete protein digestion and creating a technical dilemma. This invention aims to achieve a safe, convenient extraction method that minimizes sample loss and simultaneously ensures proteinase K activity and nucleic acid integrity through the synergistic design of a low-toxicity, high-efficiency dewaxing agent and a segmented ion protection strategy.

[0023] This invention innovatively integrates the compatibility of low-polarity fatty acid ester dewaxing agents with the time-sequential control of segmented ion protection, thereby overcoming the shortcomings of existing technologies. First, utilizing the excellent compatibility between the fatty acid esters composed of C11-C18 aliphatic and C1-C8 hydrocarbon groups in the dewaxing agent and paraffin, paraffin is rapidly dissolved under gentle heating conditions. Furthermore, because the dewaxing agent has a lower density than water, physical removal is unnecessary; it is directly added to an aqueous tissue lysis buffer to form a liquid coating layer, avoiding the multiple washings and sample loss associated with traditional xylene dewaxing. Simultaneously, its low toxicity and low volatility eliminate health and environmental risks. Subsequently, in the tissue lysis stage, a lysis buffer without metal ion chelating agents is used to provide proteinase K-dependent lysis. The highly active environment allows for efficient digestion of histones cross-linked with nucleic acids. After lysis, a reverse cross-linking protection solution containing metal ion chelating agents and monovalent salt ions is added to chelate divalent metal ions in the system, eliminating the catalytic conditions for nucleic acid hydrolysis at high temperatures, while maintaining the stability of the system's ionic strength. This design integrates the three steps of dewaxing, lysis, and cross-linking reversal into a continuous operation, resolving the contradiction between proteinase K activity and high-temperature protection of nucleic acids. It also reduces sample loss and operational risks by simplifying the process, ultimately obtaining high-purity, high-integrity nucleic acid products to meet downstream molecular detection needs.

[0024] Specifically, such as Figure 1 As shown, a method for extracting DNA / RNA from formalin-fixed paraffin-embedded tissues includes the following steps: S100. Formalin-fixed paraffin-embedded tissue section samples are mixed with a dewaxing agent and heated and incubated to fully dissolve the paraffin. The dewaxing agent contains fatty acid esters with the general formula R1-COO-R2, wherein R1 is an aliphatic hydrocarbon group of C11-C18, and the aliphatic hydrocarbon group is saturated or unsaturated, straight-chain or branched, and R2 is a hydrocarbon group of C1-C8. S200: Add tissue lysis buffer and proteinase K to S100, and perform tissue lysis at 30-70°C. The tissue lysis buffer contains buffer compounds and anionic surfactants, but does not contain metal ion chelating agents. The concentration of proteinase K is 20 mg / mL. S300. After tissue lysis is completed, a reverse cross-linking protective solution is added and mixed evenly to chelate metal ions to obtain a mixture. The reverse cross-linking protective solution contains a metal ion chelating agent. S400: The mixture obtained in S300 is incubated at 80℃-100℃ to reverse the methylene crosslinking between nucleic acids and proteins; S500. After heat incubation, the mixture is centrifuged to separate into layers. The lower aqueous phase containing DNA / RNA is retained and the upper organic phase is discarded. Then, a binding solution is added to the retained lower aqueous phase to adsorb DNA / RNA onto the solid adsorption carrier. After washing with washing solution I and washing solution II in sequence, the DNA / RNA is eluted with elution solution to obtain purified DNA / RNA.

[0025] Example 1 In this embodiment, human lung cancer FFPE tissue sections were used as samples. Isopropyl myristate containing Solvent Blue 35 indicator dye was used as a dewaxing agent. Combined with tissue lysis buffer without metal ion chelating agent and reverse crosslinking protection solution containing EDTA and KCl, DNA / RNA extraction was completed through a "one-tube" operation and a segmented ion protection strategy.

[0026] Implementation materials and reagents Sample: Human lung cancer FFPE tissue block, 5 serial sections, each 8μm thick, placed in 1.5mL enzyme-free centrifuge tubes.

[0027] Reagents: Dewaxing agent: Isopropyl myristate (IPM), add 0.005% (w / v) Solvent Blue 35 (oil-soluble indicator dye), mix thoroughly and store at 4°C away from light.

[0028] Tissue lysis buffer: 5% (w / v) N-lauroyl sarcosinate sodium, 25 mM tris(hydroxymethyl)aminomethane (TRIS), pH adjusted to 8.0 with hydrochloric acid, autoclaved and stored at room temperature.

[0029] Reversal crosslinking protection solution: 50mM disodium ethylenediaminetetraacetate (EDTA) 2Na) and 1M potassium chloride (KCl) were added, and the pH was adjusted to 8.0 with hydrochloric acid. After filtration and sterilization, the mixture was stored at 4°C.

[0030] Proteinase K: 20 mg / mL, aliquot and store at -20°C.

[0031] Binding solution: 40% (w / v) guanidine hydrochloride, 50% (v / v) isopropanol, 5% (v / v) Triton X-100, store at room temperature protected from light.

[0032] Washing solution I: 50% (w / v) guanidine hydrochloride, 0.5% (w / v) sodium citrate, 30% (v / v) ethanol, store at room temperature.

[0033] Washing solution II: 80% (v / v) ethanol, store at room temperature.

[0034] Eluent: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0, autoclaved and stored at 4°C.

[0035] Implementation steps Dewaxing and Pretreatment: Add 600 μL of dewaxing agent to the centrifuge tube containing FFPE sections, tighten the cap, and place it in a 56°C incubator for 5 minutes. During this time, vortex for 30 seconds every 2 minutes to ensure complete dissolution of paraffin. Observe the formation of two layers in the centrifuge tube: the upper layer is the blue dewaxing agent phase (containing dissolved paraffin), and the lower layer is the precipitate of tissue fragments. Figure 2 As shown in Group C, the blue dye clearly indicates the position of the organic phase, preventing accidental aspiration during subsequent operations.

[0036] Tissue lysis: Without removing the upper dewaxing agent, directly add 300 μL of tissue lysis buffer and 20 μL of proteinase K to the centrifuge tube. Vortex for 10 seconds to ensure sufficient contact between the lysis buffer and the tissue. Place the centrifuge tube in a 56°C incubator at 1000 rpm for 60 minutes, vortexing every 15 minutes to promote tissue lysis. After incubation, the solution should be clear with no obvious tissue fragments remaining.

[0037] Reverse crosslinking protection: Add 20 μL of reverse crosslinking protection solution to the lysis buffer, gently invert the centrifuge tube 5 times to mix, and avoid violent shaking that may cause nucleic acid breakage. At this time, EDTA rapidly chelates divalent metal ions in the system, and KCl maintains the stability of the ionic strength of the system.

[0038] High-temperature reversal of crosslinking: The temperature of the thermostatic mixer is raised to 90°C, and the rotation speed is maintained at 1000 rpm for 60 minutes to reverse the methylene crosslinking between nucleic acids and proteins. During incubation, the upper dewaxing agent forms a liquid coating layer, effectively preventing the evaporation of the aqueous phase and ensuring the stability of the reaction system concentration.

[0039] Phase separation: Remove the centrifuge tube and place it in a high-speed refrigerated centrifuge. Centrifuge at 12000×g for 2 minutes. After centrifugation, the tube clearly separates into three layers: the upper layer is a blue dewaxing agent-paraffin phase; the middle layer contains a very small amount of white cell debris; and the lower layer is a clear aqueous phase containing DNA / RNA, with a volume of approximately 300 μL. Carefully pipette through the upper organic phase and the middle debris layer, and aspirate 300 μL of the lower aqueous phase into a new enzyme-free centrifuge tube, avoiding aspiration of the organic phase.

[0040] Binding and Purification: Add 300 μL of binding buffer to the aspirated aqueous phase, vortex for 10 seconds, and incubate at room temperature for 5 minutes to allow DNA / RNA to fully bind with the ionizing salts in the binding buffer. Transfer the mixture to an adsorption column, centrifuge at 8000×g for 1 minute, and discard the waste liquid in the collection tube. Add 500 μL of washing buffer I to the adsorption column, centrifuge at 8000×g for 1 minute, and discard the waste liquid; then add 700 μL of washing buffer II, centrifuge at 8000×g for 1 minute, and discard the waste liquid, repeating the washing once. Place the adsorption column in a new collection tube, centrifuge at 12000×g for 2 minutes to completely remove residual washing buffer. Transfer the adsorption column to a 1.5 mL enzyme-free centrifuge tube, add 50 μL of elution buffer to the center of the adsorption membrane, incubate at room temperature for 2 minutes, centrifuge at 12000×g for 1 minute, and collect the elution liquid in the centrifuge tube, which is the purified DNA / RNA sample. Store at -20℃ for later use.

[0041] Implementation Results Nucleic acid concentration and purity: The extracted DNA / RNA concentration was 45.8 ng / μL, with an OD260 / 280 ratio of 1.89 and an OD260 / 230 ratio of 2.02, indicating that there were very few impurities such as proteins and polysaccharides remaining in the sample.

[0042] Nucleic acid integrity: Agarose gel electrophoresis results showed that the extracted DNA bands were clear with no obvious tailing, and the main fragment lengths were concentrated above 1000bp. The 28S and 18S rRNA in the RNA bands were clearly distinguishable, indicating that the nucleic acid integrity was good and no serious degradation had occurred, which could meet the requirements of long fragment sequencing.

[0043] Operational efficiency: The dewaxing stage takes only 6 minutes, and there is no need for multiple centrifugation and washing to remove the dewaxing agent. The total operation time is reduced by about 30% compared with the traditional xylene method, and no physical loss of tissue fragments or nucleic acids occurs.

[0044] In summary, this embodiment achieves efficient and safe extraction of DNA / RNA from FFPE tissues. The isopropyl myristate dewaxing agent containing Solvent Blue 35 is not only non-toxic and low in volatility, but also forms a clear interface with the aqueous phase. Combined with the "one-tube" operation, it effectively avoids the health risks and sample loss of the traditional xylene method. The segmented ion protection strategy, by delaying the addition of metal ion chelating agents, ensures the high activity of proteinase K to completely lyse the tissue, while protecting the integrity of nucleic acids during the high-temperature reversal cross-linking stage. Experimental results show that the nucleic acid extracted by this method has high concentration, excellent purity, and good integrity. The operation is convenient and efficient, providing a reliable nucleic acid sample preparation solution for clinical molecular diagnostics and basic research.

[0045] Example 2 This embodiment uses human liver cancer FFPE tissue as the research object and sets up three groups of experiments. The traditional xylene dewaxing agent (Group A), the existing improved mineral oil dewaxing agent (Group B), and the isopropyl myristate dewaxing agent of the present invention (Group C) were used respectively. DNA / RNA extraction was carried out under the same tissue lysis, reverse cross-linking and purification conditions. The dewaxing time, nucleic acid yield, purity and integrity of the three groups were compared.

[0046] Implementation materials and reagents Sample: Human liver cancer FFPE tissue block, 9 consecutive sections, each 10μm thick, randomly divided into three groups A, B and C, with 3 sections in each group, and placed in 1.5mL enzyme-free centrifuge tubes.

[0047] Reagents: Group A dewaxing agents: analytical grade xylene, anhydrous ethanol, and 70% ethanol.

[0048] Group B dewaxing agent: mineral oil (purity ≥99%).

[0049] Group C dewaxing agent: Isopropyl myristate + 0.005% (w / v) Solvent Blue 35.

[0050] The tissue lysis buffer, reverse cross-linking protection buffer, proteinase K, binding buffer, washing buffer I, washing buffer II, and elution buffer were the same as in Example 1.

[0051] Implementation steps Dewaxing and pretreatment: Group A, Xylene Method: Add 1 mL of xylene to a centrifuge tube, vortex for 30 seconds, let stand at room temperature for 5 minutes, centrifuge at 12000×g for 2 minutes, and discard the supernatant; repeat the xylene dewaxing step once. Then add 1 mL of anhydrous ethanol, vortex for 30 seconds, centrifuge at 12000×g for 2 minutes, and discard the supernatant; repeat the anhydrous ethanol washing once. Finally, add 1 mL of 70% ethanol, vortex for 30 seconds, centrifuge at 12000×g for 2 minutes, discard the supernatant, open the cap and air dry at room temperature for 10 minutes until the ethanol has completely evaporated.

[0052] Group B, Mineral Oil Method: Add 600 μL of mineral oil to a centrifuge tube, tighten the cap, and incubate in an 80°C constant temperature mixer for 5 minutes, vortexing for 30 seconds during this period to dissolve the paraffin. At this time, the mineral oil and aqueous phase can be observed to form separate layers, but the interface is not clear enough. Figure 2 As shown in Group B.

[0053] Group C, Method of the Invention: The operation is the same as the dewaxing and pretreatment steps in Example 1. After incubation at 56°C for 5 minutes, a clear blue organic phase and lower tissue precipitate are formed in the centrifuge tube, with a clear interface, as shown. Figure 2 As shown in Group C.

[0054] Tissue lysis: Group A: Add 300 μL of tissue lysis buffer and 20 μL of proteinase K to the air-dried centrifuge tubes, vortex for 10 seconds, and incubate at 56°C for 60 minutes.

[0055] Group B: No need to remove mineral oil, directly add 300μL of tissue lysis buffer and 20μL of proteinase K, vortex mix for 10 seconds, and incubate at 56℃ for 60 minutes.

[0056] Group C: The procedure is the same as the tissue lysis step in Example 1.

[0057] Reversed crosslinking protection, high-temperature reverse crosslinking, phase separation, binding and purification: all three sets of experiments were carried out according to the corresponding steps in Example 1 to ensure that all experimental conditions except for the dewaxing agent were completely consistent.

[0058] Implementation Results Performance comparison during the dewaxing stage: Group A took 15 minutes for the entire dewaxing, washing, and air-drying process. During multiple centrifugations and supernatant aspiration, a small amount of tissue fragments remained on the inner wall of the centrifuge tubes, indicating significant sample loss. Groups B and C both took 6 minutes for dewaxing, with no washing step and extremely low sample loss. Furthermore, Group A had a pungent xylene odor during operation, posing a health risk; Group B's mineral oil had high viscosity, making it easy to introduce small oil droplets when aspirating the lower aqueous phase; Group C's dewaxing agent was odorless, had low viscosity, a clear blue interface, and facilitated convenient liquid aspiration.

[0059] Nucleic acid concentration and purity: The results of the three nucleic acid extractions, as measured by a nucleic acid concentration analyzer, are shown in Table 1 below. Table 1 As shown in Table 1 above, the nucleic acid concentration in group C was significantly higher than that in groups A and B, and the OD260 / 280 and OD260 / 230 ratios were closer to the ideal range, indicating that it had fewer impurities such as protein and organic solvent residues and higher purity.

[0060] Nucleic acid integrity: Agarose gel electrophoresis results showed that the nucleic acid bands in group A had obvious tailing, with fragments concentrated below 500bp, indicating severe degradation; the nucleic acid bands in group B had less tailing than group A, but still showed some degradation; the nucleic acid bands in group C were clear and neat, with the main fragments being over 1000bp in length and without obvious tailing, indicating that its nucleic acid integrity was the best. This was due to the liquid coating layer formed by the dewaxing agent effectively preventing the evaporation of the aqueous phase and the oxidation of nucleic acids, while avoiding the influence of residual oil phase on nucleic acids.

[0061] In summary, this embodiment verifies the comprehensive advantages of the dewaxing agent of the present invention through comparative experiments. Although traditional xylene dewaxing agents can dissolve paraffin, they are highly toxic, cumbersome to operate, and result in significant sample loss, as well as poor nucleic acid extraction quality. While mineral oil dewaxing agents reduce toxicity and simplify operation, they have high viscosity, unclear interfaces, and easily lead to oil phase residue, affecting the purity and integrity of nucleic acids. The isopropyl myristate dewaxing agent of the present invention combines the characteristics of low toxicity, low viscosity, and clear interface. Combined with oil-soluble indicator dyes, it not only achieves rapid and safe dewaxing but also avoids sample loss and oil phase residue problems, significantly improving the yield, purity, and integrity of nucleic acid extraction, thus ensuring the accuracy of subsequent molecular detection.

[0062] Example 3 In this embodiment, dewaxed human colorectal cancer FFPE tissue was used as a sample. Three experimental groups were set up to verify the effectiveness of the segmented ion protection strategy: control group 1 (EDTA added throughout the process), control group 2 (no EDTA added throughout the process), and experimental group 3 (EDTA added with delay). The effects of different ion protection strategies on the extraction effect were evaluated by detecting tissue lysis effect, nucleic acid concentration, purity, and qPCR amplification efficiency.

[0063] Implementation materials and reagents Sample: Human colorectal cancer FFPE tissue block, 6 consecutive sections, each 8μm thick, after dewaxing with the isopropyl myristate dewaxing agent of this invention, were randomly divided into control group 1, control group 2 and experimental group 3, with 2 sections in each group.

[0064] Reagents: Control group 1 lysis buffer: tissue lysis buffer containing 20 mM EDTA, with the other components the same as the tissue lysis buffer in Example 1.

[0065] Control group 2 lysis buffer: Tissue lysis buffer from Example 1, without EDTA.

[0066] The lysis buffer for experimental group 3 was the same as that for control group 2; the reverse crosslinking protection solution was the same as that in Example 1, containing 50 mM EDTA.

[0067] Proteinase K, binding buffer, washing buffer I, washing buffer II, and elution buffer are the same as in Example 1.

[0068] Implementation steps Tissue lysis: Control group 1: Add 300 μL of control group 1 lysis buffer and 20 μL of proteinase K to the dewaxed sample, vortex mix for 10 seconds, and incubate at 56°C for 60 minutes.

[0069] Control group 2: Add 300 μL of control group 2 lysis buffer and 20 μL of proteinase K to the dewaxed sample, and perform the remaining operations as in control group 1.

[0070] Experimental Group 3: Add 300 μL of lysis buffer and 20 μL of proteinase K to the dewaxed sample, and perform the same procedures as control group 1. After incubation, observe the lysis status of the three groups of samples.

[0071] Reverse crosslinking treatment: Control group 1: After incubation, add 20 μL of purified water and vortex mix for 10 seconds.

[0072] Control group 2: After incubation, add 20 μL of purified water and vortex mix for 10 seconds.

[0073] Experimental group 3: After incubation, add 20 μL of reverse crosslinking protection solution and gently invert to mix 5 times.

[0074] High-temperature reverse crosslinking: All three groups of samples were incubated in a 90℃ constant temperature mixer for 60 minutes.

[0075] Phase separation, binding and purification: All three groups of samples were processed according to the corresponding steps in Example 1 to ensure a consistent purification process.

[0076] Nucleic acid quality testing: The nucleic acid concentration and OD value of the three groups of samples were measured using a nucleic acid concentration meter; the integrity of the nucleic acid was detected by agarose gel electrophoresis, and the results are as follows. Figure 3 As shown; the nucleic acid amplification efficiency was detected by real-time quantitative PCR. The reaction system was 20 μL, and the reaction conditions were: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 10 seconds, 60℃ annealing for 30 seconds, 40 cycles; melting curve analysis: 95℃ for 15 seconds, 60℃ for 1 minute, 95℃ for 15 seconds.

[0077] Implementation Results Tissue lysis status: After incubation, the solution in control group 1 was turbid, and undigested tissue fragments were visible at the bottom, indicating that proteinase K activity was inhibited by EDTA and tissue lysis was incomplete. The solutions in control group 2 and experimental group 3 were clear and transparent, with no obvious tissue fragments remaining, indicating that the lysis buffer without EDTA can ensure high proteinase K activity and achieve complete tissue lysis.

[0078] Nucleic acid concentration and purity: The test results are shown in Table 2 below: Table 2 As shown in Table 2, the nucleic acid concentration in experimental group 3 was significantly higher than that in the two control groups, and the purity index was also better. This indicates that the strategy of delaying the addition of EDTA not only ensured the tissue lysis efficiency but also reduced nucleic acid degradation and improved the extraction yield.

[0079] Nucleic acid integrity: Agarose gel electrophoresis results are as follows Figure 3As shown, the nucleic acid band in control group 1 was blurry and showed some degree of degradation; the band in control group 2 had severe tailing and obvious fragmentation of nucleic acid fragments, mainly concentrated in the 200-500bp range; the band in experimental group 3 was clear, without obvious tailing, and the fragment length was concentrated above 1000bp, with the best integrity, which confirms that EDTA in the reverse crosslinking protective solution can effectively chelate divalent metal ions and inhibit nucleic acid hydrolysis at high temperature.

[0080] qPCR amplification efficiency: The average Ct value of control group 1 was 29.5, control group 2 was 31.2, and experimental group 3 was 27.8. The lower the Ct value, the higher the nucleic acid amplification efficiency. The ΔCt value of experimental group 3 was 2.7 lower than that of control group 1 and 4.4 lower than that of control group 2, indicating that its nucleic acid had a higher effective template content and better fragment integrity, which can better meet the needs of downstream PCR detection.

[0081] In summary, this embodiment clarifies the core advantages of the segmented ion protection strategy through three sets of control experiments. Traditional methods that add EDTA throughout the entire process inhibit proteinase K activity, leading to incomplete tissue lysis and low nucleic acid yield. While EDTA-free methods ensure lysis efficiency, divalent metal ions at high temperatures catalyze nucleic acid hydrolysis, resulting in severe fragmentation. The delayed EDTA addition strategy of this invention provides a highly active, chelate-free environment for proteinase K during the lysis stage, ensuring complete tissue digestion and nucleic acid release. Adding EDTA before high-temperature reversal of cross-linking rapidly chelates metal ions, effectively protecting nucleic acid integrity. Simultaneously, monovalent salt ions maintain system stability, ultimately achieving a simultaneous improvement in nucleic acid yield, purity, and integrity. This resolves the contradiction between enzyme activity and nucleic acid protection in existing technologies, providing high-quality nucleic acid samples for downstream molecular detection.

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for extracting DNA / RNA from formalin-fixed paraffin-embedded tissues, characterized in that, The steps of this method are as follows: S100. Formalin-fixed paraffin-embedded tissue section samples are mixed with a dewaxing agent and heated and incubated to fully dissolve the paraffin. The dewaxing agent contains fatty acid esters with the general formula R1-COO-R2, wherein R1 is a C11-C18 aliphatic hydrocarbon group, and the aliphatic hydrocarbon group is saturated or unsaturated, straight-chain or branched, and R2 is a C1-C8 hydrocarbon group. S200: Add tissue lysis buffer and proteinase K to S100 and perform tissue lysis at 30-70°C. The tissue lysis buffer contains buffer compounds and anionic surfactants, but does not contain metal ion chelating agents. The concentration of proteinase K is 20 mg / mL. S300. After tissue lysis is completed, a reverse cross-linking protective solution is added and mixed evenly to chelate metal ions to obtain a mixture. The reverse cross-linking protective solution contains a metal ion chelating agent. S400: The mixture obtained in S300 is incubated at 80℃-100℃ to reverse the methylene crosslinking between nucleic acids and proteins; S500. After heat incubation, the mixture is centrifuged to separate into layers. The lower aqueous phase containing DNA / RNA is retained and the upper organic phase is discarded. Then, a binding solution is added to the retained lower aqueous phase to adsorb DNA / RNA onto the solid adsorption carrier. After washing with washing solution I and washing solution II in sequence, the DNA / RNA is eluted with elution solution to obtain purified DNA / RNA.

2. The method for extracting DNA / RNA from formalin-fixed paraffin-embedded tissue according to claim 1, characterized in that, In S100, the fatty acid ester of the dewaxing agent is selected from isopropyl myristate, 2-ethylhexyl palmitate, isopropyl palmitate, ethyl oleate, or a mixture thereof.

3. The method for extracting DNA / RNA from formalin-fixed paraffin-embedded tissue according to claim 1, characterized in that, In S100, the dewaxing agent further includes an oil-soluble indicator dye, which is selected from dyes whose chromophores are anthraquinone or azo structures, including Solvent Blue 35, Solvent Green 3, Sudan Black B, and Disperse Blue 14.

4. The method for extracting DNA / RNA from formalin-fixed paraffin-embedded tissue according to claim 1, characterized in that, In S200, the buffer compound of the tissue lysis buffer is selected from one or more of N-(tris(hydroxymethyl)methyl)glycine, tris(hydroxymethyl)aminomethane, di(2-hydroxyethyl)iminotris(hydroxymethyl)methane, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), N-cyclohexyl-2-aminoethanesulfonic acid, N,N-bis(2-hydroxyethyl)glycine, 2-(N-morpholino)ethanesulfonic acid, piperazine-1,4-bis(2-ethanesulfonic acid), 3-(N-morpholino)propanesulfonic acid, and phosphate buffer, and the concentration of the buffer compound is ≤500mM.

5. The method for extracting DNA / RNA from formalin-fixed paraffin-embedded tissue according to claim 1, characterized in that, In S200, the anionic surfactant of the tissue lysis fluid is selected from one or more of dodecyl sarcosine, sodium N-lauroyl sarcosine, sodium dodecyl sulfate, sodium dodecyl sulfonate, and sodium dodecylbenzene sulfonate, and the concentration of the anionic surfactant is 0.1%-10%.

6. The method for extracting DNA / RNA from formalin-fixed paraffin-embedded tissue according to claim 1, characterized in that, In S300, the reverse crosslinking protective liquid contains a metal ion chelating agent; The metal ion chelating agent is selected from one or more of ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid-ethylene glycol, citrate, diethylenetriaminepentaacetic acid, or aminotriacetic acid; After adding the reverse crosslinking protective liquid, the final concentration of the metal ion chelating agent in the reaction system is 0.1 mM-50 mM.

7. The method for extracting DNA / RNA from formalin-fixed paraffin-embedded tissue according to claim 1, characterized in that, In the S300, the reverse crosslinking protective liquid also contains monovalent salt ions; The monovalent salt ion is selected from one or more of lithium chloride, sodium chloride, or potassium chloride; After adding the reverse crosslinking protective solution, the final concentration of monovalent salt ions in the reaction system is 1mM-100mM.

8. The method for extracting DNA / RNA from formalin-fixed paraffin-embedded tissue according to claim 1, characterized in that, In the S500, the binding solution is a buffer solution containing a high concentration of ionizing salt, nonionic surfactant and organic solvent; The washing solution I is a buffer solution containing liquid salt, buffer and alcohol; The washing solution II is a buffer solution containing buffer and alcohol; The elution buffer is a low-salt buffer solution; The solid-phase adsorption carrier is an adsorption column or magnetic beads.

9. A method for extracting DNA / RNA from formalin-fixed paraffin-embedded tissue according to claim 1, characterized in that, During processes S100 to S400, there is no need to physically remove the dewaxing agent. Because the dewaxing agent has a density less than water, it remains stable in the upper layer of the aqueous phase after the addition of tissue lysis solution and reverse crosslinking protection solution, forming a liquid coating layer.

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