Extraction reagent, co-extraction method and kit for DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) of paraffin-embedded tissue

Abstract

The invention relates to the field of biomedical examination, and particularly discloses an extraction reagent, a co-extraction method and a kit for DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) of paraffin-embedded tissue. The invention relates to a reagent for extracting DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) of paraffin embedded tissue, which comprises a DNA binding solution containing 4-5M guanidinium isothiocyanate, 4-8wt% of Triton X-100 and 1-2wt% of polyvinylpyrrolidone, and the pH (potential of hydrogen) value of the reagent is 5.5-6.0. The invention relates to a co-extraction method of DNA and RNA of a paraffin-embedded tissue. The co-extraction method comprises the steps of digestion and lysis, DNA separation, RNA separation, cleaning and elution. The co-extraction kit for the DNA and RNA of the paraffin-embedded tissue comprises the DNA binding solution disclosed by the invention. According to the method, high-purity DNA and RNA can be effectively extracted from the same paraffin-embedded tissue at the same time, so that more accurate and comprehensive experimental data is provided for molecular pathology research.

Description

Technical Field

[0001] The invention relates to the field of biomedical testing, and in particular to an extraction reagent, a co-extraction method and a kit for DNA and RNA of paraffin-embedded tissue. Background Art

[0002] Once cells or tissues are separated from living biological samples, they often lose their activity in a short period of time. Dead cells are affected by autolysis and microbial degradation, resulting in the destruction of cell or tissue structure, and then the degradation of biological macromolecules such as proteins and nucleic acids. To prevent this from happening, the paraffin-embedded tissue (FFPE) method is currently commonly used to fix biological samples to protect the proteins and nucleic acids therein from degradation. In the process of paraffin-embedded tissue, formaldehyde is used to fix biological samples. Due to its excellent cross-linking effect, it can form covalent bonds between various biological molecules in biological samples, such as proteins and nucleic acids. Although formaldehyde can effectively fix biological samples, it will also cause cross-linking between DNA and RNA, making it difficult to extract DNA or RNA from the sample.

[0003] In order to ensure the purity of DNA or RNA extraction, traditional extraction methods usually focus on extracting DNA or RNA from paraffin-embedded tissues alone, and it is difficult to extract DNA and RNA simultaneously. In the field of molecular pathology, such as for the diagnosis or prognosis of tumor diseases, it is generally necessary to analyze the DNA and RNA of paraffin-embedded tissue samples at the same time. Current technology usually involves splitting paraffin-embedded tissue samples and extracting DNA and RNA separately for analysis. However, this mode of operation is not ideal. First, in actual operation, there are usually only a very small amount of paraffin-embedded tissue samples available, which cannot support segmentation and multiple separate purifications; secondly, the composition of paraffin-embedded tissue samples is usually uneven, and tumor cells often only exist in the matrix of healthy cells. If the samples are split, it may lead to inaccurate test results.

[0004] Therefore, it is necessary to find a method that can simultaneously extract DNA and RNA from the same paraffin-embedded tissue sample. Summary of the invention

[0005] In order to overcome the technical problem that traditional extraction methods can only extract DNA or RNA from paraffin-embedded tissues separately, the present application provides an extraction reagent, a co-extraction method and a kit for DNA and RNA from paraffin-embedded tissues, which can effectively extract high-purity DNA and RNA from the same paraffin-embedded tissue at the same time, thereby providing more accurate and comprehensive experimental data for molecular pathology research.

[0006] In the first aspect, the present application provides a paraffin-embedded tissue DNA and RNA extraction reagent using the following technical solution: A paraffin-embedded tissue DNA and RNA extraction reagent comprises a DNA binding solution and an RNA binding solution; the DNA binding solution comprises 4M-5M guanidine isothiocyanate, 4wt%-8wt% Triton X-100, and 1wt%-2wt% polyvinyl pyrrolidone, and the pH value of the DNA binding solution is adjusted to 5.5-6.0 by using sodium acetate.

[0007] In the above technical scheme, the present application can effectively promote the adsorption of DNA by magnetic beads and significantly inhibit the adsorption of RNA by selecting guanidine isothiocyanate as the ionizing agent of the DNA binding solution, and by selecting Triton X-100 and polyvinyl pyrrolidone as the surfactant of the DNA binding solution, thereby achieving efficient extraction of DNA and achieving the purpose of efficient separation of DNA and RNA. At the same time, the present application can further optimize the binding efficiency of DNA and magnetic beads and the extraction purity of DNA by adjusting the pH value of the DNA binding solution to 5.5-6.0.

[0008] Preferably, the DNA binding solution further comprises 1wt%-2wt% lecithin and 10wt%-15wt% ethanol.

[0009] In the above technical solution, the present application further adds 1wt%-2wt% lecithin and 10wt%-15wt% ethanol, which helps to stabilize the binding of DNA to magnetic beads, reduce DNA loss, and further improve the purity of DNA extraction.

[0010] Preferably, the RNA binding solution is one or a combination of isopropanol and anhydrous ethanol.

[0011] In a second aspect, the present application provides a method for co-extraction of DNA and RNA from paraffin-embedded tissues using the following technical solution: A method for co-extraction of DNA and RNA from paraffin-embedded tissues, comprising the following steps: Step 1, digestion and lysis: adding dewaxing solution to the slices of paraffin-embedded tissue to remove the paraffin, and then adding digestion solution and protease solution for digestion and lysis; Step 2, DNA separation: add magnetic beads and DNA binding solution, mix well, and separate in a magnetic field to obtain magnetic beads adsorbed with DNA and supernatant; Step 3, RNA separation: add magnetic beads and RNA binding solution to the supernatant obtained in step 2, mix well, and separate in a magnetic field to obtain magnetic beads that adsorb RNA; Step 4, cleaning: adding corresponding cleaning solutions to the magnetic beads adsorbing DNA and the magnetic beads adsorbing RNA for cleaning; Step 5, elution: adding elution solution to the washed DNA-adsorbed magnetic beads and RNA-adsorbed magnetic beads, respectively, to obtain DNA and RNA after separation; The DNA binding solution is the DNA binding solution described in the first aspect, and the RNA binding solution is the RNA binding solution described in the first aspect.

[0012] In the above technical scheme, the present application provides an efficient and simple method for co-extraction of DNA and RNA from paraffin-embedded tissues, which can not only simultaneously obtain high-purity DNA and RNA, but also is easy to operate and is suitable for the analysis of paraffin-embedded tissue samples in molecular pathology research.

[0013] Wherein, the use of dewaxing solution in step 1 is to remove paraffin in tissue sections, and the use of digestive solution and protease solution is to release DNA and RNA. The use of magnetic beads is based on its ability to bind to DNA. The use of magnetic beads and DNA binding solution in step 2 ensures the binding efficiency of DNA and magnetic beads, while inhibiting the adsorption of RNA. Through the action of the magnetic field, the magnetic beads adsorbed with DNA can be separated from the solution. The combination of RNA binding solution and magnetic beads in step 3 also ensures the efficient extraction of RNA. The cleaning solution in step 4 is to be able to effectively wash away impurities. The eluent in step 5 is to elute the DNA and RNA adsorbed on the magnetic beads separately without destroying the structure of DNA and RNA.

[0014] Preferably, the digestive solution comprises 0.1wt%-1.2wt% sodium dodecyl sulfate and 0.1wt%-0.5wt% ethylenediaminetetraacetic acid, and the pH value of the digestive solution is adjusted to 6.0-9.0 using tris(hydroxymethyl)aminomethane.

[0015] Preferably, the protease added to the protease solution is proteinase K, and the activity of proteinase K is 30-40 U / mg.

[0016] Preferably, the step 4 specifically includes: Step 4-1, DNA cleaning: Step 4-1-1, first DNA cleaning: Add 400-600uL DNA cleaning solution 1 to the magnetic beads adsorbed with DNA, vortex mix for 10-20s, transfer to the magnetic stand for adsorption for 1-1.5min, and discard the solution.

[0017] Step 4-1-2, second DNA cleaning: add 400-600uL DNA cleaning solution 2 to the magnetic beads adsorbed with DNA, vortex mix for 10-20s, transfer to the magnetic stand for adsorption for 1-1.5min, and discard the solution.

[0018] Step 4-2, RNA cleaning: Step 4-2-1, first cleaning of RNA: add 400-600uL RNA cleaning solution 1 to the magnetic beads adsorbed with RNA, vortex mix for 500-600s, transfer to the magnetic stand for adsorption for 1-1.5min, and discard the solution.

[0019] Step 4-2-2, second RNA cleaning: add 400-600uL RNA cleaning solution 2 to the magnetic beads adsorbed with RNA, vortex mix for 10-20s, transfer to the magnetic stand for adsorption for 1-1.5min, and discard the solution.

[0020] Preferably, the DNA cleaning solution 1 comprises 10wt%-15wt% ethanol, 1-3M guanidine isothiocyanate, 0.1wt%-2.5wt% tris(hydroxymethyl)aminomethane, and the pH value is adjusted to 5.0-7.0 with hydrochloric acid.

[0021] Preferably, the DNA cleaning solution 2 contains 70wt%-80wt% ethanol, 0.1wt%-2.5wt% sodium chloride, and 0.01wt%-0.1wt% tris(hydroxymethyl)aminomethane.

[0022] Preferably, the RNA cleaning solution 1 comprises 20wt%-30wt% ethanol, 0.01wt%-0.1wt% calcium chloride, 0.01wt%-0.1wt% magnesium chloride, 0.01wt%-0.1wt% tris(hydroxymethyl)aminomethane, the pH value is adjusted to 5.0-7.0 with hydrochloric acid, and 0.1-2mg / mL RNase Free DNase I is added before use.

[0023] Preferably, the RNA cleaning solution 2 contains 70wt%-80wt% ethanol, 0.1wt%-2.5wt% sodium chloride, and 0.01wt%-0.1wt% tris(hydroxymethyl)aminomethane.

[0024] Preferably, the eluent is 9-11 Mm Tris with a pH of 7.0-7.5.

[0025] Preferably, the magnetic beads are silanol-based nanomagnetic beads with a particle size of 500nm-5000nm.

[0026] In a third aspect, the present application provides a kit for co-extraction of DNA and RNA from paraffin-embedded tissues using the following technical solution: A kit for co-extraction of DNA and RNA from paraffin-embedded tissues, comprising the reagent for extracting DNA and RNA from paraffin-embedded tissues as described in the first aspect.

[0027] Preferably, the co-extraction kit for DNA and RNA from paraffin-embedded tissues specifically comprises: a dewaxing solution, a digestion solution, a protease solution, silanol-based nanomagnetic beads, a DNA binding solution, an RNA binding solution, a cleaning solution, and an elution solution.

[0028] In the above technical solution, the present application provides a kit to further facilitate the co-extraction of DNA and RNA from paraffin-embedded tissues, further simplify the operation process, ensure the efficiency and repeatability of the extraction process, reduce operation errors, and provide a powerful tool for molecular pathology research. At the same time, the kit adopts magnetic bead extraction technology, which can realize the automation of complex extraction processes.

[0029] In summary, the present application includes at least one of the following beneficial technical effects: 1. This application can simultaneously extract DNA and RNA from the same paraffin-embedded tissue sample, reducing the use of rare samples and improving the accuracy of molecular pathology research or diagnosis.

[0030] 2. This application improves the extraction efficiency and purity of DNA and RNA in paraffin-embedded tissues, simplifies the extraction operation process, can automate the operation process, reduce experimental errors, and provide an efficient, simple and reliable tool for molecular pathology research. DETAILED DESCRIPTION

[0031] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0032] Example 1 A method for co-extraction of DNA and RNA from paraffin-embedded tissues, comprising the following steps: Step 1, digestion and lysis: Take 3 20um paraffin-embedded tissue sections, add 0.8mL dewaxing solution to remove the paraffin, then add 0.25mL digestion solution and 0.02mL proteinase K solution, incubate at 55℃ for 60min, incubate at 90℃ for 60min, centrifuge and take 0.2mL digestion solution into a new centrifuge tube.

[0033] Step 2, DNA separation: Add 20 μL of magnetic bead solution and 0.3 mL of DNA binding solution to the centrifuge tube, invert and mix for 4 minutes, then transfer to the magnetic stand and separate the magnetic beads adsorbed with DNA and the supernatant with the magnetic field.

[0034] Step 3, RNA separation: Add 20 μL of magnetic bead solution and 0.3 mL of RNA binding solution to the supernatant obtained in step 2, mix by inversion for 2 minutes, and then transfer to a magnetic stand to separate the magnetic beads that adsorb RNA with the magnetic field.

[0035] Step 4-1, DNA cleaning: Step 4-1-1, first DNA cleaning: Add 500uL DNA cleaning solution 1 to the magnetic beads adsorbed with DNA, vortex mix for 15s, transfer to the magnetic stand for adsorption for 1min, and discard the solution.

[0036] Step 4-1-2, second DNA cleaning: add 500uL DNA cleaning solution 2 to the magnetic beads adsorbed with DNA, vortex mix for 15s, transfer to the magnetic stand for adsorption for 1min, and discard the solution.

[0037] Step 4-2, RNA cleaning: Step 4-2-1, first RNA cleaning: add 500uL RNA cleaning solution 1 to the magnetic beads adsorbed with RNA, vortex mix for 600s, transfer to the magnetic stand for adsorption for 1min, and discard the solution.

[0038] Step 4-2-2, second RNA cleaning: add 500uL RNA cleaning solution 2 to the magnetic beads adsorbed with RNA, vortex mix for 15s, transfer to the magnetic stand for adsorption for 1min, and discard the solution.

[0039] Step 5, elution: absorb the residual liquid from the washed DNA-adsorbed magnetic beads and RNA-adsorbed magnetic beads, air-dry for 5 minutes, add 50 μL of elution solution respectively, oscillate at 55°C for 5 minutes, then transfer to a magnetic stand and place for 1 minute. After separation, DNA and RNA are extracted respectively.

[0040] The dewaxing liquid is prepared as follows: 60 wt% tetradecane, 12 wt% pentadecane, and 8 wt% turpentine.

[0041] The digestive solution is prepared as follows: 1.0 wt% sodium dodecyl sulfate and 0.2 wt% ethylenediaminetetraacetic acid, and the pH value is adjusted to 7.0 using tris(hydroxymethyl)aminomethane.

[0042] The proteinase K solution is prepared as follows: 40 wt% glycerol, 0.4 wt% calcium chloride, and tris(hydroxymethyl)aminomethane is used to adjust the pH value to 7.5.

[0043] Among them, the activity of proteinase K is 30U / mg.

[0044] The magnetic beads are silanol-based nano-magnetic beads, and the particle size of the silanol-based nano-magnetic beads is between 500nm and 5000nm.

[0045] The magnetic bead solution was prepared as follows: 4 wt % sodium chloride, and the pH value was adjusted to 7.5 using tris(hydroxymethyl)aminomethane.

[0046] The DNA binding solution is prepared as follows: 4M guanidine isothiocyanate, 4wt% Triton X-100, 2wt% polyvinyl pyrrolidone, and the pH value is adjusted to 5.5 with sodium acetate.

[0047] Among them, the RNA binding solution is isopropanol.

[0048] The DNA cleaning solution 1 contains 12 wt % ethanol, 2 M guanidine isothiocyanate, and 1.2 wt % tris(hydroxymethyl)aminomethane, and the pH value is adjusted to 6.0 with hydrochloric acid.

[0049] The DNA cleaning solution 2 contains 75 wt % ethanol, 1.2 wt % sodium chloride, and 0.05 wt % tris(hydroxymethyl)aminomethane.

[0050] The RNA cleaning solution 1 contains 25 wt% ethanol, 0.05 wt% calcium chloride, 0.05 wt% magnesium chloride, and 0.05 wt% tris(hydroxymethyl)aminomethane. The pH value is adjusted to 6.0 with hydrochloric acid, and 1 mg / mL RNase Free DNase I is added before use.

[0051] The RNA cleaning solution 2 contains 75 wt % ethanol, 1.5 wt % sodium chloride, and 0.05 wt % tris(hydroxymethyl)aminomethane.

[0052] The elution buffer was RNase-Free 10 mM Tris with a pH of 7.4.

[0053] Example 2 A method for co-extraction of DNA and RNA from paraffin-embedded tissue, which differs from Example 1 in that it comprises the following steps: Step 1, digestion and lysis: Take 3 20um paraffin-embedded tissue sections, add 1mL dewaxing solution to remove the paraffin, then add 0.3mL digestion solution and 0.03mL proteinase K solution, incubate at 55℃ for 50min, incubate at 90℃ for 55min, centrifuge and take 0.2mL digestion solution into a new centrifuge tube.

[0054] Step 2, DNA separation: Add 15 μL of magnetic bead solution and 0.3 mL of DNA binding solution to the centrifuge tube, invert and mix for 3 minutes, then transfer to the magnetic rack and separate the magnetic beads and supernatant with DNA adsorbed by the magnetic field.

[0055] Step 3, RNA separation: Add 15 μL of magnetic bead solution and 0.3 mL of RNA binding solution to the supernatant obtained in step 2, mix by inversion for 2 minutes, and then transfer to a magnetic stand to separate the magnetic beads that adsorb RNA with the magnetic field.

[0056] Step 4-1, DNA cleaning: Step 4-1-1, first DNA cleaning: Add 400uL DNA cleaning solution 1 to the magnetic beads adsorbed with DNA, vortex mix for 20s, transfer to the magnetic stand for adsorption for 1min, and discard the solution.

[0057] Step 4-1-2, second DNA cleaning: add 400uL DNA cleaning solution 2 to the magnetic beads adsorbed with DNA, vortex mix for 20s, transfer to the magnetic stand for adsorption for 1min, and discard the solution.

[0058] Step 4-2, RNA cleaning: Step 4-2-1, first RNA cleaning: Add 400uL RNA cleaning solution 1 to the magnetic beads adsorbed with RNA, vortex mix for 600s, transfer to the magnetic stand for adsorption for 1min, and discard the solution.

[0059] Step 4-2-2, second RNA cleaning: add 400uL RNA cleaning solution 2 to the magnetic beads adsorbed with RNA, vortex mix for 20s, transfer to the magnetic stand for adsorption for 1min, and discard the solution.

[0060] Step 5, elution: absorb the residual liquid from the washed DNA-adsorbed magnetic beads and RNA-adsorbed magnetic beads, air-dry for 4 minutes, add 45 μL of elution solution respectively, oscillate at 55°C for 4 minutes, then transfer to a magnetic rack and place for 1 minute. After separation, DNA and RNA are extracted respectively.

[0061] The dewaxing liquid is prepared as follows: 80 wt% tetradecane, 10 wt% pentadecane, and 10 wt% turpentine.

[0062] The digestive solution is prepared as follows: 0.9 wt % sodium dodecyl sulfate and 0.5 wt % ethylenediaminetetraacetic acid, and the pH value is adjusted to 8.0 using tris(hydroxymethyl)aminomethane.

[0063] The proteinase K solution is prepared as follows: 30 wt% glycerol, 0.5 wt% calcium chloride, and tris(hydroxymethyl)aminomethane is used to adjust the pH value to 7.0.

[0064] Among them, the activity of proteinase K is 40U / mg.

[0065] The magnetic beads are silanol-based nano-magnetic beads, and the particle size of the silanol-based nano-magnetic beads is between 500nm and 5000nm.

[0066] The magnetic bead solution was prepared as follows: 3 wt% sodium chloride, and the pH value was adjusted to 8.0 using tris(hydroxymethyl)aminomethane.

[0067] The DNA binding solution was prepared as follows: 4 M guanidine isothiocyanate, 8 wt % Triton X-100, 1 wt % polyvinyl pyrrolidone, and the pH value was adjusted to 5.5 with sodium acetate.

[0068] Among them, the RNA binding solution is isopropanol.

[0069] The DNA cleaning solution 1 contains 10 wt % ethanol, 3 M guanidine isothiocyanate, and 1.0 wt % tris(hydroxymethyl)aminomethane, and the pH value is adjusted to 5.0 with hydrochloric acid.

[0070] The DNA cleaning solution 2 contains 80 wt % ethanol, 1 wt % sodium chloride, and 0.1 wt % tris(hydroxymethyl)aminomethane.

[0071] The RNA cleaning solution 1 contains 20 wt % ethanol, 0.01 wt % calcium chloride, 0.1 wt % magnesium chloride, and 0.01 wt % tris(hydroxymethyl)aminomethane. The pH value is adjusted to 7.0 with hydrochloric acid, and 2 mg / mL RNase Free DNase I is added before use.

[0072] The RNA cleaning solution 2 contains 80 wt % ethanol, 1.0 wt % sodium chloride, and 0.1 wt % tris(hydroxymethyl)aminomethane.

[0073] The elution buffer was RNase-Free 10 mM Tris with a pH of 7.4.

[0074] Example 3 A method for co-extraction of DNA and RNA from paraffin-embedded tissue, which differs from Example 1 in that it comprises the following steps: Step 1, digestion and lysis: Take 3 20um paraffin-embedded tissue sections, add 0.8mL dewaxing solution to remove the paraffin, then add 0.20mL digestion solution and 0.02mL proteinase K solution, incubate at 55℃ for 65min, incubate at 90℃ for 65min, centrifuge and take 0.2mL digestion solution into a new centrifuge tube.

[0075] Step 2, DNA separation: Add 25 μL of magnetic bead solution and 0.3 mL of DNA binding solution to the centrifuge tube, invert and mix for 6 minutes, then transfer to the magnetic stand and separate the magnetic beads adsorbed with DNA and the supernatant with the magnetic field.

[0076] Step 3, RNA separation: Add 25 μL of magnetic bead solution and 0.3 mL of RNA binding solution to the supernatant obtained in step 2, mix by inversion for 3 minutes, and then transfer to a magnetic stand to separate the magnetic beads that adsorb RNA with the magnetic field.

[0077] Step 4-1, DNA cleaning: Step 4-1-1, first DNA cleaning: Add 600uL DNA cleaning solution 1 to the magnetic beads adsorbed with DNA, vortex mix for 10s, transfer to the magnetic stand for adsorption for 1.5min, and discard the solution.

[0078] Step 4-1-2, second DNA cleaning: add 600uL DNA cleaning solution 2 to the magnetic beads adsorbed with DNA, vortex mix for 10s, transfer to the magnetic stand for adsorption for 1.5min, and discard the solution.

[0079] Step 4-2, RNA cleaning: Step 4-2-1, first RNA cleaning: add 600uL RNA cleaning solution 1 to the magnetic beads adsorbed with RNA, vortex mix for 500s, transfer to the magnetic stand for adsorption for 1.5min, and discard the solution.

[0080] Step 4-2-2, second RNA cleaning: add 600uL RNA cleaning solution 2 to the magnetic beads adsorbed with RNA, vortex mix for 10s, transfer to the magnetic stand for adsorption for 1.5min, and discard the solution.

[0081] Step 5, elution: absorb the residual liquid from the washed DNA-adsorbed magnetic beads and RNA-adsorbed magnetic beads, air-dry for 5 minutes, add 55 μL of elution solution respectively, oscillate at 55°C for 6 minutes, then transfer to a magnetic rack and place for 1 minute. After separation, DNA and RNA are extracted respectively.

[0082] The dewaxing liquid is prepared as follows: 50wt% tetradecane, 15wt% pentadecane, and 5wt% turpentine.

[0083] The digestive solution is prepared as follows: 1.2 wt% sodium dodecyl sulfate and 0.1 wt% ethylenediaminetetraacetic acid, and the pH value is adjusted to 6.0 using tris(hydroxymethyl)aminomethane.

[0084] The proteinase K solution is prepared as follows: 50 wt% glycerol, 0.1 wt% calcium chloride, and tris(hydroxymethyl)aminomethane is used to adjust the pH value to 7.0.

[0085] Among them, the activity of proteinase K is 30U / mg.

[0086] The magnetic beads are silanol-based nano-magnetic beads, and the particle size of the silanol-based nano-magnetic beads is between 500nm and 5000nm.

[0087] The magnetic bead solution was prepared as follows: 5 wt% sodium chloride, and the pH value was adjusted to 7.0 using tris(hydroxymethyl)aminomethane.

[0088] The DNA binding solution is prepared as follows: 5M guanidine isothiocyanate, 4wt% Triton X-100, 2wt% polyvinyl pyrrolidone, and the pH value is adjusted to 6.0 with sodium acetate.

[0089] Among them, the RNA binding solution is isopropanol.

[0090] The DNA cleaning solution 1 contains 15 wt % ethanol, 1 M guanidine isothiocyanate, and 2.5 wt % tris(hydroxymethyl)aminomethane, and the pH value is adjusted to 7.0 with hydrochloric acid.

[0091] The DNA cleaning solution 2 contains 70 wt % ethanol, 2.5 wt % sodium chloride, and 0.01 wt % tris(hydroxymethyl)aminomethane.

[0092] The RNA cleaning solution 1 contains 30 wt % ethanol, 0.1 wt % calcium chloride, 0.01 wt % magnesium chloride, and 0.1 wt % tris(hydroxymethyl)aminomethane. The pH value is adjusted to 5.0 with hydrochloric acid, and 0.1 mg / mL RNase Free DNase I is added before use.

[0093] The RNA cleaning solution 2 contains 70 wt % ethanol, 2.5 wt % sodium chloride, and 0.01 wt % tris(hydroxymethyl)aminomethane.

[0094] The elution buffer was RNase-Free 10 mM Tris with a pH of 7.4.

[0095] Example 4 A method for co-extracting DNA and RNA from paraffin-embedded tissues. The difference from Example 1 is that the DNA binding solution also contains 1wt% lecithin and 10wt% ethanol.

[0096] Example 5 A method for co-extracting DNA and RNA from paraffin-embedded tissues. The difference from Example 1 is that the DNA binding solution also contains 2wt% lecithin and 15wt% ethanol.

[0097] Comparative Example 1 A method for co-extracting DNA and RNA from paraffin-embedded tissues. The difference from Example 1 is that the pH value of the DNA binding solution is adjusted to 4.0 or 5.0 using sodium acetate.

[0098] Comparative Example 2 A method for co-extracting DNA and RNA from paraffin-embedded tissues, which is different from Example 1 in that an equal amount of guanidine isothiocyanate in the DNA binding solution is replaced by guanidine hydrochloride.

[0099] Comparative Example 3 A method for co-extracting DNA and RNA from paraffin-embedded tissues. The difference from Example 1 is that Triton X-100 in the DNA binding solution is replaced by polyethylene glycol 400 in equal amounts.

[0100] Comparative Example 4 A method for co-extracting DNA and RNA from paraffin-embedded tissues. The difference from Example 1 is that polyvinyl pyrrolidone in the DNA binding solution is replaced by polyethylene glycol 400 in equal amounts.

[0101] Comparative Example 1 DNA was extracted using a commercially available magnetic bead method kit for extracting DNA from paraffin-embedded tissues, and the purity of the extracted DNA was tested and compared with the concentration and purity of the DNA extracted in Example 1 of the present application.

[0102] Comparative Example 2 RNA was extracted using a commercially available magnetic bead method kit for extracting RNA from paraffin-embedded tissues, and the purity of the extracted RNA was tested and compared with the concentration and purity of the RNA extracted in Example 1 of the present application.

[0103] Detection: Various sample detections were performed on the DNA and RNA of the above-mentioned embodiments, comparative examples and control examples.

[0104] Detection method: Use the UV spectrophotometer Nanodrop 21000 to measure the A260 / A280 and A260 / A230 values. When the A260 / A280 of DNA is around 1.6-1.8, the purity is high, and when the A260 / A230 of RNA is around 1.9-2.0, the purity is high. The test results are shown in Table 1-10.

[0105] Table 1: Test results of animal liver and kidney extracted by the method of Example 1 Table 2: Test results of animal liver and kidney extracted by the method of Example 2 Table 3: Test results of animal liver and kidney extracted by the method of Example 3 Table 4: Test results of animal liver and kidney extracted by the method of Example 4 Table 5: Test results of animal liver and kidney extracted by the method of Example 5 Table 6: Changing the pH value of the DNA binding solution, referring to the method of Example 1, comparing the test results of extracting animal liver and kidney under the conditions of pH 4.0, 5.0, 5.5, and 6.0 of the DNA binding solution Table 7: Comparative Example 1 and Comparative Example 2 respectively extracted animal liver and kidney test results Table 8: Comparative Example 1 and Comparative Example 3, respectively, for extracting the test results of animal liver and kidney Table 9: Comparative Example 1 and Comparative Example 3, respectively, for extracting the test results of animal liver and kidney Table 10: Comparative results of animal liver and kidney extraction using the methods of Example 1, Comparative Example 1 and Comparative Example 2 Combined with the analysis of the above test results, we can see that the DNA and RNA in the sample are extracted using the DNA and RNA co-extraction method of the embodiment of the present application, which can ensure the stability of the extraction results and ensure that the extracted DNA and RNA are maintained at a high purity and concentration.

[0106] Specifically combined with the test results of Table 6, the extraction effect of DNA and RNA is best when the pH of the DNA binding solution is 5.5-6.0. Within this pH range, the concentration and yield of DNA and RNA are both high, and the purity indexes A260 / A280 and A260 / A230 are both within the ideal range, indicating that the quality of the extracted nucleic acid is high. By further comparing the experimental data of Example 1 with Comparative Example 2, Comparative Example 3 and Comparative Example 4, i.e., Tables 7-9, it can be found that Example 1 has good stability and repeatability when extracting DNA and RNA from animal liver and kidney, and the quality of the extracted nucleic acid is high. In addition, further combined with the test results of Table 10, when analyzing the experimental data of Comparative Example 1 with Comparative Example 1 and Comparative Example 2, we can see that the purity index of Example 1 and the extraction concentration are all within a good range, which shows that the simultaneous extraction of DNA and RNA using the method of Example 1 is stable and reliable.

[0107] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A reagent for extracting DNA and RNA from paraffin-embedded tissue, characterized in that: It comprises a DNA binding solution and an RNA binding solution; the DNA binding solution comprises 4M-5M guanidine isothiocyanate, 4wt%-8wt% Triton X-100, and 1wt%-2wt% polyvinyl pyrrolidone, and the pH value of the DNA binding solution is adjusted to 5.5-6.0 by using sodium acetate.

2. A paraffin-embedded tissue DNA and RNA extraction reagent according to claim 1, characterized in that: The DNA binding solution also contains 1wt%-2wt% lecithin and 10wt%-15wt% ethanol.

3. A paraffin-embedded tissue DNA and RNA extraction reagent according to claim 1, characterized in that: The RNA binding solution is one or a combination of isopropanol and anhydrous ethanol.

4. A method for co-extraction of DNA and RNA from paraffin-embedded tissue, characterized in that: The following steps are involved: Step 1, digestion and lysis: adding dewaxing solution to the slices of paraffin-embedded tissue to remove the paraffin, and then adding digestion solution and protease solution for digestion and lysis; Step 2, DNA separation: add magnetic beads and DNA binding solution, mix well, and separate in a magnetic field to obtain magnetic beads adsorbed with DNA and supernatant; Step 3, RNA separation: add magnetic beads and RNA binding solution to the supernatant obtained in step 2, mix well, and separate in a magnetic field to obtain magnetic beads that adsorb RNA; Step 4, cleaning: adding corresponding cleaning solutions to the magnetic beads adsorbing DNA and the magnetic beads adsorbing RNA for cleaning; Step 5, elution: adding elution solution to the washed DNA-adsorbed magnetic beads and RNA-adsorbed magnetic beads, respectively, to obtain DNA and RNA after separation; The DNA binding solution is the DNA binding solution as described in any one of claims 1 to 3, and the RNA binding solution is the RNA binding solution as described in any one of claims 1 to 3.

5. A method for co-extraction of DNA and RNA from paraffin-embedded tissue according to claim 4, characterized in that: The digestion solution contains 0.1wt%-1.2wt% sodium dodecyl sulfate and 0.1wt%-0.5wt% ethylenediaminetetraacetic acid, and the pH value of the digestion solution is adjusted to 6.0-9.0 by using tris(hydroxymethyl)aminomethane.

6. The method for co-extraction of DNA and RNA from paraffin-embedded tissue according to claim 4, characterized in that: The protease added to the protease solution is proteinase K, and the activity of the proteinase K is 30-40 U / mg.

7. The method for co-extraction of DNA and RNA from paraffin-embedded tissue according to claim 4, characterized in that: The eluent is 9-11 Mm Tris, pH 7.0-7.

5.

8. The method for co-extraction of DNA and RNA from paraffin-embedded tissue according to claim 4, characterized in that: The magnetic beads are silanol-based nanomagnetic beads with a particle size of 500nm-5000nm.

9. A kit for co-extraction of DNA and RNA from paraffin-embedded tissues, characterized in that: The invention comprises a paraffin-embedded tissue DNA and RNA extraction reagent as described in any one of claims 1 to 3.

10. A kit for co-extraction of DNA and RNA from paraffin-embedded tissues according to claim 9, characterized in that: Specifically include: Dewaxing solution, digestion solution, protease solution, silanol-based nanomagnetic beads, DNA binding solution, RNA binding solution, cleaning solution, and elution solution.

Images