A column-based RNA extraction method

Abstract

The present invention provides a column-based RNA extraction method, comprising the following steps: ① adding a lysate to a cell sample or tissue sample for lysing; ② adding anhydrous ethanol to the lysed cell sample or tissue sample, blowing and sucking, mixing evenly, transferring the above solution to an adsorption column, and placing the adsorption column in a collection tube, centrifuging, and pouring out the liquid in the collection tube; ③ adding a DNA enzyme and an elution buffer mixture to the adsorption column, standing; ④ adding a washing buffer to the adsorption column, centrifuging, and pouring out the waste liquid in the collection tube; putting the adsorption column back into the collection tube, centrifuging again, and pouring out the waste liquid in the collection tube; ⑤ transferring the adsorption column to a clean centrifuge tube, opening the cover and drying, and obtaining an adsorption film; ⑥ adding an elution buffer to the center of the adsorption film, standing; centrifuging, obtaining an RNA solution and preserving. The lysate, washing buffer, and elution buffer of the present invention use environmentally friendly and safe ingredients, and the extracted RNA has high concentration and high purity.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an RNA extraction method based on a column method. Background Art

[0002] RNA, the full name of ribonucleic acid, is an important molecule in organisms. Together with DNA, or deoxyribonucleic acid, it constitutes the genetic material basis of living organisms. Unlike DNA, RNA is mainly present in the cytoplasm and plays an important role in transmitting genetic information and guiding protein synthesis in cells. In biomedical research, RNA extraction is an important experimental step. By extracting RNA from cells or tissues, the expression of specific genes can be studied, thereby gaining a deep understanding of the molecular mechanisms of life activities. In the process of RNA extraction, cells or tissues must first be broken to release RNA. Subsequently, through a series of chemical and physical treatment steps, proteins, DNA and other impurities are removed to finally obtain pure RNA. One of the existing RNA extraction methods is the column method. However, the lysis solution and elution buffer used in the existing column method mostly contain toxic substances such as phenol and chloroform, which not only bring dangers to the operation of the experiment, but also bring additional management and organizational costs to the laboratory, and are also prone to sample contamination. Therefore, the research on environmentally friendly and safe lysis solutions and elution buffers is particularly important. Summary of the invention

[0003] In view of this, the present invention proposes a column-based RNA extraction method to solve the above problems.

[0004] The technical solution of the present invention is achieved as follows: a column-based RNA extraction method comprising the following steps:

[0005] ① Add lysis buffer to cell samples or tissue samples for lysis;

[0006] ② Add anhydrous ethanol to the lysed cell sample or tissue sample, pipette and aspirate 9-11 times to mix evenly, transfer the above solution to the adsorption column, and place the adsorption column in the collection tube, centrifuge and discard the liquid in the collection tube;

[0007] ③ Add DNA enzyme and elution buffer mixture to the adsorption column and let it stand for 4-6 minutes;

[0008] ④ Add washing buffer to the adsorption column, centrifuge, and discard the waste liquid in the collection tube; put the adsorption column back into the collection tube, centrifuge again, and discard the waste liquid in the collection tube;

[0009] ⑤ Transfer the adsorption column into a clean 1.5 mL centrifuge tube, open the cover and dry it to obtain the adsorption film;

[0010] ⑥ Add elution buffer to the center of the adsorption membrane and let it stand for 2-3 minutes; centrifuge to obtain RNA solution and store it at -75~-85℃;

[0011] The lysate is composed of tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, 1-ethyl-3-methylimidazole acetate, saponin and water in a mass volume ratio of g / mL of (1.0-2.0):(0.1-0.5):(0.5-2.0):(0.05-0.2):(10.0-15.0).

[0012] Furthermore, in step ①, the cell samples include adherent cell samples and suspended cell samples, and the tissue samples include visceral tissue samples and tumor tissue samples. The adherent cell samples are lysed according to the following method: 1×10 5 -2×10 6 Add 1×10 adherent cells to a 1.5 mL centrifuge tube to obtain an adherent cell sample, add phosphate buffer solution to the adherent cell sample for washing, dry the phosphate buffer solution, add 450-550 μL lysis solution, place in a shaker at 23-25° C. and shake at 120-150 r / min for 1.5-2.5 min; the suspended cell sample is lysed according to the following method: take 1×10 6 -3×10 6 Place the suspended cells in a 1.5 mL centrifuge tube to obtain a suspended cell sample, centrifuge at 400-600 r / min for 2-4 min, discard the supernatant, add 450-550 μL of lysis solution to the centrifuge tube, blow 9-11 times with a pipette, and then vortex mix at 1500-2500 r / min for 8-12 s; the visceral tissue sample and tumor tissue sample are lysed according to the following method: weigh 1-5 mg of visceral tissue or 1-5 mg of tumor tissue and add it to a 1.5 mL centrifuge tube. tube, obtain visceral tissue samples or tumor tissue samples, add 450-550 μL lysis solution to the above tissue samples, homogenize with a grinding rod or homogenizer, vortex mix at 1500-2500 r / min for 8-12 s at 23-25°C, let stand for 4-6 min, vortex mix at 2000-3000 r / min for 8-12 s, and then centrifuge at 10000-15000 r / min for 1.5-2.5 min, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0013] Furthermore, in step ②, the volume ratio of anhydrous ethanol to cell sample or tissue sample is (0.9-1.1):1.0, and centrifugation is performed at 10000-15000 r / min for 0.5-1.5 min.

[0014] Furthermore, in step ③, the amount of DNA enzyme added is 1.5-2.5 μL, and the amount of elution buffer added is 8-12 μL, and the DNA enzyme and the elution buffer are mixed to obtain a DNA enzyme elution buffer mixture.

[0015] Furthermore, in step ④, the amount of washing buffer added is 450-550 μL, the centrifugal separation speed is 10000-15000 r / min, the time is 0.5-1.5 min, and the centrifugal separation speed is again 10000-15000 r / min, and the time is 0.5-1.5 min.

[0016] Furthermore, in step ④, the washing buffer solution is composed of tris(hydroxymethyl)aminomethane hydrochloride, magnesium chloride, Tween 20 and water in a mass volume ratio of g / mL of (0.5-1.0):(0.3-0.5):(0.01-0.1):(6.0-8.0).

[0017] Furthermore, the drying in step ⑤ is air-drying, the air-drying wind speed is 3-5m / s, and the air-drying time is 2-3min.

[0018] Furthermore, in step ⑥, the amount of elution buffer added is 20-30 μL, the centrifugal separation speed is 10000-15000 r / min, and the time is 0.5-1.5 min.

[0019] Furthermore, the elution buffer is composed of tris(hydroxymethyl)aminomethane hydrochloride), arginine, cyclodextrin, sodium chloride, sodium dodecyl sulfate and water in a mass volume ratio of g / mL of (0.1-1):(0.01-0.1):(0.05-0.5):(4.0-6.0):(0.05-0.5):(40.0-50.0).

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention uses an environmentally friendly and safe lysate and elution buffer for RNA extraction. The lysate in the present invention is composed of tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, 1-ethyl-3-methylimidazole acetate, saponin, and water in a specific ratio, and the elution buffer is composed of tris(hydroxymethyl)aminomethane hydrochloride, arginine, cyclodextrin, sodium chloride, sodium dodecyl sulfate, and water in a specific ratio. The tris(hydroxymethyl)aminomethane hydrochloride in the lysate ensures that the lysate is within the stable pH range of RNA, thereby protecting the integrity of RNA. Ethylenediaminetetraacetic acid can effectively reduce the activity of RNase, thereby preventing RNA from being degraded during the extraction process, and ensuring the purity and integrity of the extracted RNA. 1-ethyl-3-methylimidazole acetate can react with the lipid bilayer of the cell membrane, destroy the integrity of the membrane, thereby more effectively releasing RNA in the cell and increasing the total concentration of RNA. Saponin can reduce the surface tension of the solution, help to better dissolve and extract RNA, enhance cell lysis, and thus increase the total concentration of RNA. The tris(hydroxymethyl)aminomethane hydrochloride) in the elution buffer maintains the pH value of the eluent to be stable, ensuring that the RNA is in a suitable environment during the elution process, and avoiding damage to the RNA caused by changes in pH. Arginine can enhance the stability and solubility of RNA, and at the same time help to separate RNA from the column material. Cyclodextrin can increase the solubility of RNA. Sodium chloride affects the binding force between RNA and the column material by adjusting the ionic strength, and promotes the elution of RNA from the carrier. The different components in the lysate and elution buffer in the present invention act synergistically through different mechanisms, so that the total concentration of RNA extracted by the method of the present invention is high and the purity is high. DETAILED DESCRIPTION

[0021] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0022] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0023] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0024] Example 1

[0025] A column-based RNA extraction method comprises the following steps:

[0026] ① Take 5×10 5The adherent cells were added into a 1.5 mL centrifuge tube to obtain an adherent cell sample, a phosphate buffer solution was added to the adherent cell sample for washing, the phosphate buffer solution was aspirated, 450 μL of lysis solution was added, and the sample was placed in a shaker and shaken at 120 r / min for 2.5 min at 23° C.; wherein the lysis solution consisted of tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, 1-ethyl-3-methylimidazole acetate, saponin, and water in a mass volume ratio of g / mL of 1.0:0.1:0.5:0.05:10.0.

[0027] ② Add anhydrous ethanol to the lysed cell sample in a volume ratio of 0.9:1.0. Pipet and draw 9 times to mix evenly. Transfer the above solution to the adsorption column, and place the adsorption column in a collection tube. Centrifuge at 10,000 r / min for 1.5 min, and discard the liquid in the collection tube.

[0028] ③ Add 1.5 μL of DNA enzyme and 8 μL of elution buffer mixture to the adsorption column to obtain a DNA enzyme elution buffer mixture, and let it stand for 4 minutes;

[0029] ④ Add 450 μL of washing buffer to the adsorption column, centrifuge at 10,000 r / min for 1.5 min, and discard the waste liquid in the collection tube; put the adsorption column back into the collection tube, centrifuge again at 10,000 r / min for 1.5 min, and discard the waste liquid in the collection tube; the washing buffer consists of tris(hydroxymethyl)aminomethane hydrochloride, magnesium chloride, Tween 20, and water with a mass volume ratio of g / mL of 0.5:0.3:0.01:6.0.

[0030] ⑤ Transfer the adsorption column into a clean 1.5mL centrifuge tube, open the lid, and air-dry at a wind speed of 3m / s for 3min to obtain an adsorption film.

[0031] ⑥ Add 20 μL of elution buffer to the center of the adsorption membrane and let it stand for 2 minutes; centrifuge at 10,000 r / min for 1.5 minutes to obtain an RNA solution and store it at -75°C; the elution buffer is composed of tris(hydroxymethyl)aminomethane hydrochloride, arginine, cyclodextrin, sodium chloride, sodium dodecyl sulfate, and water in a mass volume ratio of 0.1:0.01:0.05:4.0:0.05:40.0 g / mL.

[0032] Example 2

[0033] A column-based RNA extraction method comprises the following steps:

[0034] ① Take 3×10 6Place each suspended cell in a 1.5 mL centrifuge tube to obtain a suspended cell sample, centrifuge at 600 r / min for 2 min, discard the supernatant, add 550 μL of lysis solution to the centrifuge tube, blow 11 times with a pipette, and then vortex mix at 2500 r / min for 8 s. The lysis solution consists of tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, 1-ethyl-3-methylimidazole acetate, saponin, and water in a mass volume ratio of g / mL of 2.0:0.5:2.0:0.2:15.0.

[0035] ② Add anhydrous ethanol to the lysed cell sample in a volume ratio of 1.1:1.0. Pipet and draw 11 times to mix evenly. Transfer the above solution to the adsorption column, and place the adsorption column in a collection tube. Centrifuge at 15,000 r / min for 0.5 min, and discard the liquid in the collection tube.

[0036] ③ Add 2.5 μL of DNA enzyme and 12 μL of elution buffer mixture to the adsorption column to obtain a DNA enzyme elution buffer mixture, and let it stand for 6 minutes;

[0037] ④ Add 550 μL of washing buffer to the adsorption column, centrifuge at 15000 r / min for 0.5 min, and discard the waste liquid in the collection tube; put the adsorption column back into the collection tube, centrifuge at 15000 r / min for 0.5 min again, and discard the waste liquid in the collection tube; the washing buffer is composed of tris(hydroxymethyl)aminomethane hydrochloride, magnesium chloride, Tween 20, and water in a mass volume ratio of 1.0:0.5:0.1:8.0 g / mL.

[0038] ⑤ Transfer the adsorption column into a clean 1.5mL centrifuge tube, open the lid, and air-dry at a wind speed of 5m / s for 2min to obtain an adsorption film.

[0039] ⑥ Add 30 μL of elution buffer to the center of the adsorption membrane and let it stand for 3 minutes; centrifuge at 15000 r / min for 0.5 min to obtain RNA solution and store it at -85°C; the elution buffer is composed of tris(hydroxymethyl)aminomethane hydrochloride, arginine, cyclodextrin, sodium chloride, sodium dodecyl sulfate, and water in a mass volume ratio of 1.0:0.1:0.5:6.0:0.5:50.0 g / mL.

[0040] Example 3

[0041] A column-based RNA extraction method comprises the following steps:

[0042] ① Weigh 2.5 mg of visceral tissue and add it to a 1.5 mL centrifuge tube to obtain a visceral tissue sample. Add 500 μL of lysis solution to the above tissue sample, homogenize with a homogenizer, vortex mix at 2000 r / min for 10 seconds at 24°C, let stand for 5 minutes, vortex mix at 2500 r / min for 10 seconds, centrifuge at 12000 r / min for 2.0 minutes, and transfer the supernatant to a new 1.5 mL centrifuge tube. The lysis solution is composed of tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, 1-ethyl-3-methylimidazole acetate, saponin, and water in a mass volume ratio of 1.5:0.3:1.2:0.12:12.5 g / mL.

[0043] ② Add anhydrous ethanol to the lysed tissue sample in a volume ratio of 1.0:1.0. Pipet and draw 10 times to mix evenly. Transfer the above solution to the adsorption column, and place the adsorption column in a collection tube. Centrifuge at 12000r / min for 1.0min, and discard the liquid in the collection tube.

[0044] ③ Add 2.0 μL of DNA enzyme and 10 μL of elution buffer mixture to the adsorption column to obtain a DNA enzyme elution buffer mixture, and let it stand for 5 minutes;

[0045] ④ Add 500 μL of washing buffer to the adsorption column, centrifuge at 12000 r / min for 1.0 min, and discard the waste liquid in the collection tube; put the adsorption column back into the collection tube, centrifuge at 12000 r / min for 1.0 min again, and discard the waste liquid in the collection tube; wherein the washing buffer is composed of tris(hydroxymethyl)aminomethane hydrochloride, magnesium chloride, Tween 20, and water with a mass volume ratio of g / mL of 0.8:0.4:0.06:7.0.

[0046] ⑤ Transfer the adsorption column into a clean 1.5mL centrifuge tube, open the lid, and air-dry at a wind speed of 4m / s for 2.5min to obtain an adsorption film.

[0047] ⑥ Add 25 μL of elution buffer to the center of the adsorption membrane and let it stand for 2.5 min; centrifuge at 12000 r / min for 1.0 min to obtain RNA solution and store it at -80°C; the elution buffer is composed of tris(hydroxymethyl)aminomethane hydrochloride, arginine, cyclodextrin, sodium chloride, sodium dodecyl sulfate, and water with a mass volume ratio of g / mL of 0.6:0.05:0.3:5.0:0.3:45.0.

[0048] Example 4

[0049] A column-based RNA extraction method comprises the following steps:

[0050] ① Weigh 1 mg of tumor tissue and add it to a 1.5 mL centrifuge tube to obtain a tumor tissue sample. Add 450 μL of lysis solution to the above tissue sample, homogenize with a homogenizer, vortex mix at 2500 r / min for 8 seconds at 25°C, let stand for 6 minutes, vortex mix at 3000 r / min for 8 seconds, and then centrifuge at 15000 r / min for 1.5 minutes. Take the supernatant and transfer it to a new 1.5 mL centrifuge tube. The lysis solution is composed of tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, 1-ethyl-3-methylimidazole acetate, saponin, and water in a mass volume ratio of 1.0:0.1:0.5:0.05:10.0 g / mL.

[0051] ② Add anhydrous ethanol to the lysed tissue sample in a volume ratio of 0.9:1.0. Pipet and draw 11 times to mix evenly. Transfer the above solution to the adsorption column, and place the adsorption column in a collection tube. Centrifuge at 15,000 r / min for 0.5 min, and discard the liquid in the collection tube.

[0052] ③ Add 1.5 μL of DNA enzyme and 8 μL of elution buffer mixture to the adsorption column to obtain a DNA enzyme elution buffer mixture, and let it stand for 6 minutes;

[0053] ④ Add 450 μL of washing buffer to the adsorption column, centrifuge at 15000 r / min for 0.5 min, and discard the waste liquid in the collection tube; put the adsorption column back into the collection tube, centrifuge at 15000 r / min for 0.5 min again, and discard the waste liquid in the collection tube; the washing buffer is composed of tris(hydroxymethyl)aminomethane hydrochloride, magnesium chloride, Tween 20, and water with a mass volume ratio of g / mL of 0.5:0.3:0.01:6.0.

[0054] ⑤ Transfer the adsorption column into a clean 1.5 mL centrifuge tube, open the lid, and air-dry at a wind speed of 5 m / s for 2 min to obtain an adsorption film.

[0055] ⑥ Add 20 μL of elution buffer to the center of the adsorption membrane and let it stand for 3 minutes; centrifuge at 15000 r / min for 0.5 min to obtain RNA solution and store it at -85°C; the elution buffer is composed of tris(hydroxymethyl)aminomethane hydrochloride, arginine, cyclodextrin, sodium chloride, sodium dodecyl sulfate and water with a mass volume ratio of g / mL of 0.1:0.01:0.05:4.0:0.05:40.0.

[0056] Example 5

[0057] A column-based RNA extraction method comprises the following steps:

[0058] ① Weigh 5 mg of tumor tissue and add it to a 1.5 mL centrifuge tube to obtain a tumor tissue sample. Add 550 μL of lysis solution to the above tissue sample, homogenize with a homogenizer, vortex mix at 1500 r / min for 12 seconds at 23°C, let stand for 4 minutes, vortex mix at 2000 r / min for 12 seconds, centrifuge at 10000 r / min for 2.5 minutes, and transfer the supernatant to a new 1.5 mL centrifuge tube. The lysis solution is composed of tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, 1-ethyl-3-methylimidazole acetate, saponin, and water in a mass volume ratio of 2.0:0.5:2.0:0.2:15.0 g / mL.

[0059] ② Add anhydrous ethanol to the lysed tissue sample in a volume ratio of 1.1:1.0. Pipet and draw 9 times to mix evenly. Transfer the above solution to the adsorption column, and place the adsorption column in a collection tube. Centrifuge at 10,000 r / min for 1.5 min, and discard the liquid in the collection tube.

[0060] ③ Add 2.5 μL of DNA enzyme and 12 μL of elution buffer mixture to the adsorption column to obtain a DNA enzyme elution buffer mixture, and let it stand for 4 minutes;

[0061] ④ Add 550 μL of washing buffer to the adsorption column, centrifuge at 10,000 r / min for 1.5 min, and discard the waste liquid in the collection tube; put the adsorption column back into the collection tube, centrifuge again at 10,000 r / min for 0.5-1.5 min, and discard the waste liquid in the collection tube; the washing buffer is composed of tris(hydroxymethyl)aminomethane hydrochloride, magnesium chloride, Tween 20, and water in a mass volume ratio of 1.0:0.5:0.1:8.0 g / mL.

[0062] ⑤ Transfer the adsorption column into a clean 1.5mL centrifuge tube, open the lid, and air-dry at a wind speed of 3m / s for 3min to obtain an adsorption film.

[0063] ⑥ Add 30 μL of elution buffer to the center of the adsorption membrane and let it stand for 2 minutes; centrifuge at 10,000 r / min for 1.5 minutes to obtain an RNA solution and store it at -75°C; the elution buffer is composed of tris(hydroxymethyl)aminomethane hydrochloride, arginine, cyclodextrin, sodium chloride, sodium dodecyl sulfate, and water in a mass volume ratio of 1.0:0.1:0.5:6.0:0.5:50.0 g / mL.

[0064] Comparative Example 1

[0065] This comparative example is compared with Example 3, except that the lysate consists of tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, 1-ethyl-3-methylimidazole acetate, saponin and water in a mass volume ratio of 2.3:0.8:0.4:0.3:16.0 in g / mL.

[0066] Comparative Example 2

[0067] This comparative example is compared with Example 3, except that the lysate is composed of tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, and water in a mass volume ratio of g / mL of 1.5:0.3:12.5.

[0068] Comparative Example 3

[0069] The difference between this comparative example and Example 3 is that the lysate consists of guanidine thiocyanate, phenol, chloroform and isopropanol in a mass volume ratio of 5:1.5:1.5:2 in g / mL.

[0070] Comparative Example 4

[0071] This comparative example is compared with Example 3, except that the elution buffer consists of tris(hydroxymethyl)aminomethane hydrochloride, arginine, cyclodextrin, sodium chloride, sodium dodecyl sulfate and water in a mass volume ratio of 1.2:0.2:0.6:7.0:0.6:55.0 g / mL.

[0072] Comparative Example 5

[0073] This comparative example is compared with Example 3, except that the elution buffer is composed of tris(hydroxymethyl)aminomethane hydrochloride, sodium chloride, sodium dodecyl sulfate and water in a mass volume ratio of 0.6:5.0:0.3:45.0 g / mL.

[0074] Comparative Example 6

[0075] This comparative example is compared with Example 3, except that the elution buffer is composed of tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, phenol and anhydrous ethanol in a mass volume ratio of 0.4:0.03:0.4:35.0 g / mL.

[0076] 1. Determination of total RNA concentration and purity

[0077] The total concentration and purity of the RNA extracted from Examples 1-5 and Comparative Examples 1-6 were measured by a spectrophotometer, wherein the purity was measured by OD260 / 280 and OD260 / 230, each of which was measured 5 times and the average values ​​were calculated and recorded in Table 1.

[0078] Table 1

[0079]

[0080]

[0081] It can be seen from Table 1 that the total concentration of RNA extracted by the method of the present invention is high, and the OD260 / 280 and OD260 / 230 of the extracted RNA are both close to 2.0, which shows that the RNA extracted by the method of the present invention has high purity, and Example 3 has better effect.

[0082] By comparing Example 3 with Comparative Example 1, the total concentration and purity of Example 3 are higher than those of Comparative Example 1. This is because the ratio of each component in the lysate needs to be precisely controlled to achieve the best cell lysis effect and RNA protection effect. Improper ratios will lead to low lysis efficiency, resulting in low total RNA concentration and low purity.

[0083] By comparing Example 3 with Comparative Example 2, the total concentration and purity of Example 3 are higher than those of Comparative Example 2. This is because the 1-ethyl-3-methylimidazole acetate lacking in the lysate of Comparative Example 2 can react with the lipid bilayer of the cell membrane, destroy the integrity of the membrane, and thus more effectively release the RNA in the cell. The saponin therein can reduce the surface tension of the solution, help to better dissolve and extract RNA, and enhance cell lysis. The total RNA concentration is increased by the action of 1-ethyl-3-methylimidazole acetate and saponin. At the same time, 1-ethyl-3-methylimidazole acetate and saponin help to effectively separate RNA from protein and DNA, thereby improving RNA purity.

[0084] By comparing Example 3 with Comparative Example 3, the total concentration and purity of Example 3 are higher than those of Comparative Example 3. This is because the phenol in Comparative Example 3 may bind to part of the RNA to form an insoluble complex, which remains in the organic phase and cannot be completely extracted into the aqueous phase, resulting in RNA loss and decreased purity. Chloroform causes RNA to be distributed between the two phases, part of which will be redistributed into the organic phase, resulting in a decrease in the RNA concentration in the aqueous phase, thereby resulting in RNA loss and decreased purity.

[0085] By comparing Example 3 with Comparative Example 4, the total concentration and purity of Example 3 are higher than those of Comparative Example 4. This is because each component needs to work synergistically at a specific ratio to ensure effective elution and purification of RNA, so that RNA with high concentration and high purity can be extracted.

[0086] By comparing Example 3 with Comparative Example 5, the total concentration and purity of Example 3 are higher than those of Comparative Example 5. This is because arginine helps to maintain the stability of the RNA structure and prevents degradation during elution and purification by interacting with RNA molecules, thereby improving the purity of RNA. At the same time, arginine also helps to separate RNA from column materials, thereby increasing the concentration of RNA. Cyclodextrin has a unique cavity structure and can form inclusion compounds, which helps to increase the solubility of RNA in the aqueous phase, thereby increasing the total concentration of RNA in the eluent. Cyclodextrin can also interact specifically with impurities, thereby achieving selective separation of RNA from impurities and further improving the purity of RNA. Therefore, the total concentration and purity of RNA extracted in Example 3 are higher than those in Comparative Example 5.

[0087] By comparing Example 3 with Comparative Example 6, the total concentration and purity of Example 3 are higher than those of Comparative Example 6. This is because the phenol in Comparative Example 6 combines with part of the RNA to form a complex, making it more difficult to effectively separate the RNA during the elution process, thereby reducing the total concentration and purity of the RNA.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A column-based RNA extraction method, characterized in that: The following steps are involved: ① Add lysis buffer to cell samples or tissue samples for lysis; ② Add anhydrous ethanol to the lysed cell sample or tissue sample, pipette and aspirate 9-11 times, mix evenly to form a solution, transfer the solution to the adsorption column, place the adsorption column in the collection tube, centrifuge, and discard the liquid in the collection tube; ③ Add DNA enzyme and elution buffer mixture to the adsorption column and let it stand for 4-6 minutes; the amount of DNA enzyme added is 1.5-2.5 μL, and the amount of elution buffer added is 8-12 μL, and the DNA enzyme and the elution buffer are mixed to obtain a DNA enzyme elution buffer mixture; the elution buffer is composed of tris(hydroxymethyl)aminomethane hydrochloride, arginine, cyclodextrin, sodium chloride, sodium dodecyl sulfate, and water in a mass volume ratio of g / mL of (0.1-1):(0.01-0.1):(0.05-0.5):(4.0-6.0):(0.05-0.5):(40.0-50.0); ④ Add washing buffer to the adsorption column, centrifuge and discard the waste liquid in the collection tube; Put the adsorption column back into the collection tube, centrifuge again, and discard the waste liquid in the collection tube; The amount of the washing buffer added is 450-550 μL, the centrifugal separation speed is 10000-15000 r / min, the time is 0.5-1.5 min, and the centrifugal separation speed is 10000-15000 r / min, and the time is 0.5-1.5 min; ⑤ Transfer the adsorption column into a clean 1.5 mL centrifuge tube, open the cover and dry it to obtain the adsorption film; ⑥ Add elution buffer to the center of the adsorption membrane and let it stand for 2-3 minutes; Centrifuge to obtain RNA solution and store it at -75 to -85°C; the amount of elution buffer added is 20-30 μL, the centrifugal speed is 10000-15000 r / min, and the time is 0.5-1.5 min; The lysate is composed of tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, 1-ethyl-3-methylimidazole acetate, saponin and water in a mass volume ratio of g / mL of (1.0-2.0):(0.1-0.5):(0.5-2.0):(0.05-0.2):(10.0-15.0).

2. The RNA extraction method based on the column method according to claim 1, characterized in that: In step ①, the cell samples include adherent cell samples and suspended cell samples, and the tissue samples include visceral tissue samples and tumor tissue samples. The adherent cell samples are lysed according to the following method: 1×10 5 -2×10 6 Add 1×10 adherent cells to a 1.5 mL centrifuge tube to obtain an adherent cell sample, add phosphate buffer solution to the adherent cell sample for washing, dry the phosphate buffer solution, add 450-550 μL lysis solution, place in a shaker at 23-25° C. and shake at 120-150 r / min for 1.5-2.5 min; the suspended cell sample is lysed according to the following method: take 1×10 6 -3×10 6 Place the suspended cells in a 1.5 mL centrifuge tube to obtain a suspended cell sample, centrifuge at 400-600 r / min for 2-4 min, discard the supernatant, add 450-550 μL of lysis solution to the centrifuge tube, blow 9-11 times with a pipette, and then vortex mix at 1500-2500 r / min for 8-12 s; the visceral tissue sample and tumor tissue sample are lysed according to the following method: weigh 1-5 mg of visceral tissue or 1-5 mg of tumor tissue and add it to a 1.5 mL centrifuge tube. tube, obtain visceral tissue samples or tumor tissue samples, add 450-550 μL lysis solution to the above tissue samples, homogenize with a grinding rod or homogenizer, vortex mix at 1500-2500 r / min for 8-12 s at 23-25°C, let stand for 4-6 min, vortex mix at 2000-3000 r / min for 8-12 s, and then centrifuge at 10000-15000 r / min for 1.5-2.5 min, and transfer the supernatant to a new 1.5 mL centrifuge tube.

3. The RNA extraction method based on the column method according to claim 1, characterized in that: In step ②, the volume ratio of anhydrous ethanol to cell sample or tissue sample is (0.9-1.1):1.0, and centrifugation is performed at 10000-15000 r / min for 0.5-1.5 min.

4. The RNA extraction method based on the column method according to claim 1, characterized in that: The drying in step ⑤ is air drying, the air drying speed is 3-5 m / s, and the air drying time is 2-3 min.

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