A method for extracting RNA from cartilage tissue

Abstract

The present invention belongs to the technical field of molecular biology detection, and discloses a method for extracting RNA from cartilage tissue. The method comprises the following steps: 1) incubating cartilage tissue powder, CTAB lysis solution, β-mercaptoethanol and proteinase K; 2) performing solid-liquid separation, taking the supernatant, and mixing it with phenol-chloroform; 3) performing solid-liquid separation, taking the supernatant, and mixing it with ethanol; 4) performing solid-liquid separation, taking the precipitate, and washing; 5) performing solid-liquid separation, taking the precipitate, and mixing it with a solvent to obtain the RNA. The RNA dissociation solution of the present invention enables RNA to be released as much as possible from mucin in the peripheral matrix of bone tissue during the lysis process through the synergistic action of CTAB, 2-mercaptoethanol and proteinase K, then uses phenol-chloroform to separate nucleic acid from protein impurities, then takes the supernatant, uses absolute ethanol to precipitate RNA, and utilizes the property of dissolving most impurities to obtain high-quality total RNA.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology detection, and specifically relates to a method for extracting RNA from cartilage tissue. Background Art

[0002] Bone tissue is hard, with a low density of bone cells, and contains a large amount of mucin (proteoglycan) and RNA in the peripheral matrix, making it difficult to separate. Traditional extraction methods cannot extract high-quality total RNA. However, with the rapid development of molecular biology, research such as genome sequencing and genetic analysis has also been widely applied and promoted. These often require high-purity and high-integrity RNA, thus having higher requirements for the quality of RNA.

[0003] Currently, methods for extracting total RNA include guanidine isothiocyanate / phenol method (trizol method) (CN107699559A), SDS method, CTAB method (CN104017804A), and extraction kits (CN106434633A), etc. However, due to the particularity of bone samples, it is difficult to obtain RNA with high purity, high yield, good integrity, and low salt ion concentration using general extraction methods, especially RNA with high purity and good integrity.

[0004] Currently, the failure to effectively separate and purify RNA in bone tissue has hindered the research progress in its molecular biology aspect. Therefore, it is very necessary to optimize and improve the RNA extraction method according to the characteristics of bone tissue to find a relatively simple, rapid, general, and efficient method for extracting total RNA from bone tissue. Summary of the Invention

[0005] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a method for extracting RNA from cartilage tissue in order to solve the problems existing in the prior art.

[0006] To achieve the above purpose, the present invention specifically adopts the following technical solutions.

[0007] The first aspect of the present invention protects a method for extracting RNA from cartilage tissue, including the following steps:

[0008] 1) Incubate cartilage tissue powder, CTAB lysis solution, β-mercaptoethanol, and proteinase K;

[0009] 2) Separate the solid and liquid, take the supernatant, and mix it with phenol-chloroform;

[0010] 3) Separate the solid and liquid, take the supernatant, and mix it with ethanol;

[0011] 4) Separate the solid and liquid, take the precipitate, and wash it;

[0012] 5) Solid-liquid separation, take the precipitate, mix it with a solvent to obtain the RNA as described above.

[0013] In certain embodiments of the present invention, the CTAB lysis solution contains 0.5% - 4 wt% of CTAB, 0.5 - 4.5 M of NaCl, 50 - 400 mM of Tris-HCl, and 50 - 200 mM of EDTA. In the present invention, Tris-HCl provides a buffer environment to prevent nucleic acids from being damaged and is beneficial to the stability of RNA; EDTA can chelate Mg 2+ and Mn 2+ , inhibit the activity of RNase and prevent RNA from being degraded; 0.5 - 4.5 M NaCl provides a high-salt environment to fully dissolve RNP (RNA-CTAB complex) and exist in the liquid phase; CTAB can dissolve the cell membrane and bind to nucleic acids to facilitate the separation of nucleic acids. Nucleic acids are soluble in high-salt solutions.

[0014] In certain specific embodiments of the present invention, the CTAB lysis solution may contain 0.5% - 1.2 wt% of CTAB, or may contain 1% - 2.5 wt% of CTAB, or may contain 2.3% - 4 wt% of CTAB. In a preferred embodiment, it contains 2 wt% of CTAB.

[0015] In certain specific embodiments of the present invention, the CTAB lysis solution may contain 0.5 - 1.8 M of NaCl, or may contain 1.5 - 3.6 M of NaCl, or may contain 3.3 - 4.5 M of NaCl. In a preferred embodiment, it contains 1.5 M of NaCl.

[0016] In certain specific embodiments of the present invention, the CTAB lysis solution may contain 50 - 180 mM of Tris-HCl, or may contain 150 - 350 M of Tris-HCl, or may contain 300 - 400 M of Tris-HCl. In a preferred embodiment, it contains 200 M of Tris-HCl.

[0017] In certain specific embodiments of the present invention, the CTAB lysis solution may contain 50 - 100 mM of EDTA, or may contain 80 - 150 mM of EDTA, or may contain 100 - 200 mM of EDTA. In a preferred embodiment, it contains 100 mM of EDTA.

[0018] In a preferred embodiment of the present invention, the CTAB lysis solution contains 2 wt% of CTAB, 1.5 M of NaCl, 200 mM of Tris-HCl, and 100 mM of EDTA.

[0019] In certain embodiments of the present invention, the pH value of the CTAB lysis solution is 7.0-9.0.

[0020] In certain specific embodiments of the present invention, the pH value of the CTAB lysis solution may be 7.0-8.2, 7.5-8.6, or 8.4-9.0. In a preferred embodiment, it is 8.0.

[0021] In certain embodiments of the present invention, the mass-to-volume ratio of the cartilage tissue powder to the CTAB lysate is 1 mg: (1-20) μL.

[0022] In certain embodiments of the present invention, the mass-to-volume ratio of the cartilage tissue powder to the CTAB lysis solution can be 1 mg: (1-9) μL, 1 mg: (8-15) μL, or 1 mg: (12-20) μL. In a preferred embodiment, the mass-to-volume ratio is 1 mg: 10 μL.

[0023] In certain embodiments of the present invention, the mass ratio of the cartilage tissue powder to proteinase K is 100 mg: (0.0001-0.002) U. Proteinase K in the present invention can digest mucin in the matrix surrounding bone tissue, assist in cleavage, and release RNA wrapped by mucin.

[0024] In certain embodiments of the present invention, the ratio of the cartilage tissue powder to proteinase K can be 100 mg: (0.0001-0.001) U, 100 mg: (0.0005-0.0015) U, or 100 mg: (0.0014-0.002) U. In a preferred embodiment, the ratio is 1 mg: 0.0004 U.

[0025] In certain embodiments of the present invention, the volume ratio of the CTAB lysis solution to β-mercaptoethanol is 100:(0.2-5). β-mercaptoethanol in the present invention can act as an antioxidant to effectively prevent phenol from being oxidized to quinone, thus avoiding browning and making phenol easier to remove.

[0026] In certain embodiments of the present invention, the volume ratio of the CTAB lysis solution to β-mercaptoethanol is 100:(0.2-1.6), or 100:(1.2-3.4), or 100:(2.2-5). In a preferred embodiment, it is 100:2.

[0027] In certain embodiments of the present invention, in 1), the incubation temperature is 40-70°C.

[0028] In certain specific embodiments of the present invention, 1), the incubation temperature can be 40 - 55°C, or 50 - 60°C, or 58 - 70°C. In a preferred embodiment, it is 50°C, 55°C, or 60°C.

[0029] In certain embodiments of the present invention, 1), the incubation time is 5 - 30 min.

[0030] In certain specific embodiments of the present invention, 1), the incubation time can be 5 - 15 min, or 10 - 19 min, or 18 - 30 min. In a preferred embodiment, it is 10 min, 15 min, and 20 min.

[0031] In certain embodiments of the present invention, 2), the phenol - chloroform is a mixture of water - saturated phenol and chloroform.

[0032] In certain specific embodiments of the present invention, the volume ratio of the water - saturated phenol to chloroform is (0.5 - 1.5):1.

[0033] In certain more specific embodiments of the present invention, the volume ratio of the water - saturated phenol to chloroform can be (0.5 - 0.9):1, or (0.8 - 1.3):1, or (0.7 - 1.5):1. In a preferred embodiment, it is 1:1.

[0034] In certain embodiments of the present invention, 2), the volume ratio of the phenol - chloroform to the supernatant is (0.5 - 2):1.

[0035] In certain specific embodiments of the present invention, 2), the volume ratio of the phenol - chloroform to the supernatant is (0.5 - 1.1):1, or (0.8 - 1.4):1, or (1.2 - 2):1. In a preferred embodiment, it is 1:1.

[0036] In certain embodiments of the present invention, 2), the solid - liquid separation is centrifugation. The conditions for centrifugation are: 1 - 6°C, 10000 - 20000 g, and centrifugation for 1 - 5 min.

[0037] In certain specific embodiments of the present invention, the conditions for centrifugation are: 3 - 5°C, 13000 - 17000 g, and centrifugation for 1 - 3 min. In a preferred embodiment, it is 4°C, 15000 g, and centrifugation for 2 min.

[0038] In certain embodiments of the present invention, 3), the volume ratio of the ethanol to the supernatant is (0.5 - 1.5):1.

[0039] In certain specific embodiments of the present invention, in 3), the volume ratio of the ethanol to the supernatant can be (0.5 - 0.9):1, or can be (0.8 - 1.2):1, or can be (0.9 - 1.5):1. In a preferred embodiment, it is 0.8:1.

[0040] In certain specific embodiments of the present invention, in 3), the ethanol is anhydrous ethanol.

[0041] In certain embodiments of the present invention, in 3), the solid-liquid separation is centrifugation. The conditions for the centrifugation are: 1 - 6°C, 10000 - 20000g, and centrifugation for 10 - 25 min.

[0042] In certain specific embodiments of the present invention, the conditions for the centrifugation are: 3 - 5°C, 13000 - 17000g, and centrifugation for 10 - 20 min. In a preferred embodiment, it is 4°C, 14000g, and centrifugation for 15 min.

[0043] In certain embodiments of the present invention, in 4), the washing is performed using an ethanol aqueous solution.

[0044] In certain specific embodiments of the present invention, the concentration of the ethanol aqueous solution is 50 - 80 v / v%. Preferably, the concentration of the ethanol aqueous solution can be 50 - 62 v / v%, or can be 58 - 73 v / v%, or can be 58 - 80 v / v%. In a preferred embodiment, it is 75 v / v%.

[0045] In certain specific embodiments of the present invention, the number of washing times is 1 - 5. Preferably, the number of times can be 1 - 3, or can be 1 - 4, or can be 1 - 5. In a preferred embodiment, it is 2 times.

[0046] In certain embodiments of the present invention, in 4), the solid-liquid separation is centrifugation. The conditions for the centrifugation are: 1 - 6°C, 10000 - 20000g, and centrifugation for 5 - 20 min.

[0047] In certain specific embodiments of the present invention, the conditions for the centrifugation are: 3 - 5°C, 13000 - 17000g, and centrifugation for 10 - 20 min. In a preferred embodiment, it is 4°C, 14000g, and centrifugation for 10 min.

[0048] In certain embodiments of the present invention, in 5), the solvent is a diethyl pyrocarbonate aqueous solution.

[0049] In certain embodiments of the present invention, the concentration of the diethyl pyrocarbonate aqueous solution is 0.05 to 0.2 wt%. Preferably, the concentration of the diethyl pyrocarbonate aqueous solution can be 0.05 to 0.1 wt%, or can be 0.07 to 0.15 wt%, or can be 0.1 to 0.2 wt%. In a preferred embodiment, it is 0.1 wt%.

[0050] In certain embodiments of the present invention, in 5), the solid-liquid separation is centrifugation. The conditions for the centrifugation are: 1 to 6 °C, 10000 to 20000 g, and centrifugation for 5 to 20 min.

[0051] In certain specific embodiments of the present invention, the conditions for the centrifugation are: 3 to 5 °C, 13000 to 17000 g, and centrifugation for 5 to 15 min. In a preferred embodiment, it is 4 °C, 15000 g, and centrifugation for 10 min.

[0052] In certain embodiments of the present invention, the particle size of the cartilage tissue powder is 0.05 to 0.5 μm. Preferably, the particle size of the cartilage tissue powder can be 0.05 to 0.12 μm, or can be 0.1 to 0.3 μm, or can be 0.2 to 0.5 μm. In a preferred embodiment, it is 0.1 μm.

[0053] In certain embodiments of the present invention, the cartilage tissue is one or more of mouse cartilage tissue, rat cartilage tissue, rabbit cartilage tissue, chicken cartilage tissue, coral tissue, and shrimp antenna tissue.

[0054] In certain embodiments of the present invention, the cartilage tissue powder is obtained by grinding the cartilage tissue. The grinding frequency is 50 to 60 Hz, and the grinding time is 15 to 25 s. Preferably, a high-throughput tissue grinder is used for grinding.

[0055] In the prior art, the CTAB method utilizes the cationic detergent CTAB to precipitate nucleic acids and acidic polysaccharides from a solution with a low sodium ion strength. Under such conditions, proteins and neutral polysaccharides remain in the solution while nucleic acids precipitate, thereby separating RNA. The present invention utilizes the property that in a solution with a high sodium ion strength, CTAB forms complexes with proteins and polysaccharides but cannot precipitate nucleic acids. A CTAB lysis solution, proteinase K, and β-mercaptoethanol are used in combination to digest and lyse cartilage tissue, prompting the rupture of chondrocytes, enzymatically digesting the histone bound to nucleic acids, fully separating RNA, and protecting the released RNA during lysis; phenol-chloroform is used to remove residual phenol and protein contamination, improving the purity of RNA; anhydrous ethanol is used to dehydrate RNA, thereby precipitating RNA. As a result, the obtained RNA has integrity, a single electrophoretic band without trailing, and high purity, and can be directly used for PCR reactions.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] (1) The present invention provides a method for improving the traditional CTAB, obtaining an extraction method suitable for total RNA of cartilage tissue, which lays a foundation for further molecular experiments and high-throughput sequencing.

[0058] (2) The present invention utilizes the synergistic effect of CTAB, proteinase K, and β-mercaptoethanol in the lysis solution to separate mucin (proteoglycan) from RNA, and removes it as an impurity during the extraction process. Then, anhydrous ethanol is used to deprive the water molecules around RNA, dehydrating RNA and making it easy to polymerize and precipitate, so that some impurities such as proteins, polysaccharides, phenols, and salts remain in the solution and do not precipitate with RNA, extracting relatively ideal total RNA, thereby improving the yield and integrity of RNA.

[0059] (3) The present invention optimizes the RNA extraction process to obtain higher-quality RNA, solves the pretreatment problems for subsequent molecular experiments, qPCR experiments, NGS sequencing and other related experiments, solves technical problems, and greatly improves the success rate of subsequent experiments. Description of the Drawings

[0060] Figure 1 Shown is the electrophoretogram of RNA obtained by the extraction method of the present invention in 1.5% agarose gel, with mouse cartilage tissue, rat cartilage tissue, rabbit cartilage tissue, chicken cartilage tissue, coral tissue, and shrimp antenna tissue as objects. Among them, M is the marker; 1.1 is the electrophoretic band with mouse cartilage tissue as the object; 2.1 is the electrophoretic band with rat cartilage tissue as the object; 3.1 is the electrophoretic band with rabbit cartilage tissue as the object; 4.1 is the electrophoretic band with chicken cartilage tissue as the object; 5.1 is the electrophoretic band with coral tissue as the object; 6.1 is the electrophoretic band with shrimp antenna tissue as the object.

[0061] Figure 2 Shown are the electrophoresis patterns of RNAs extracted by the Trizol method from mouse cartilage tissue, rat cartilage tissue, rabbit cartilage tissue, chicken cartilage tissue, coral tissue, and shrimp antenna tissue as objects in the present invention in a 1.5% agarose gel. Among them, 1.2 is the band corresponding to mouse cartilage tissue; 2.2 is the band corresponding to rat cartilage tissue; 3.2 is the band corresponding to rabbit cartilage tissue; 4.2 is the band corresponding to chicken cartilage tissue; 5.2 is the band corresponding to coral tissue; 6.2 is the band corresponding to shrimp antenna tissue.

[0062] Figure 3 Shown are the electrophoresis patterns of RNAs extracted using the EASYspin Plus Bone Tissue RNA kit from mouse cartilage tissue, rat cartilage tissue, rabbit cartilage tissue, chicken cartilage tissue, coral tissue, and shrimp antenna tissue as objects in the present invention in a 1.5% agarose gel. Among them, 1.3 is the band corresponding to mouse cartilage tissue; 2.3 is the band corresponding to rat cartilage tissue; 3.3 is the band corresponding to rabbit cartilage tissue; 4.3 is the band corresponding to chicken cartilage tissue; 5.3 is the band corresponding to coral tissue; 6.3 is the band corresponding to shrimp antenna tissue.

[0063] Figure 4 Shown are the electrophoresis patterns of RNAs extracted by the extraction method of the present invention from crab hepatopancreas tissue and iris root tissue as objects in Examples 7 and 8 of the present invention in a 1.5% agarose gel. Among them, 7 is the band corresponding to crab hepatopancreas tissue; 8 is the band corresponding to iris root tissue. Detailed implementation manners

[0064] The applicant has discovered through long-term research that CTAB lysis buffer, proteinase K, and β-mercaptoethanol can cooperate to separate mucin (proteoglycan) and RNA in bone tissue, and as the extraction process progresses, the mucin is removed as an impurity; then anhydrous ethanol is used to deprive the water molecules around the RNA, causing the RNA to lose water and be prone to polymerization and precipitation, while impurities such as proteins, polysaccharides, phenols, and salts remain in the solution, thereby extracting high-purity and high-integrity RNA.

[0065] Taking cartilage tissue as the object, comparing with the Trizol method and commercial bone tissue extraction kits, it is found that the RNA obtained by the extraction method of the present invention has good integrity and high concentration; in addition, taking non-cartilage tissue and plant tissue as objects respectively, the RNA obtained by the method of the present invention has poor integrity or low concentration. Therefore, the extraction method of the present invention for extracting RNA from cartilage tissue can obtain high-purity and good-integrity RNA.

[0066] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0067] Before further describing the specific implementation manners of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific implementation manners described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific implementation manners, rather than limiting the protection scope of the present invention. For the test methods without specific conditions indicated in the following embodiments, they are generally carried out under conventional conditions or according to the conditions recommended by each manufacturer.

[0068] When the embodiments give numerical ranges, it should be understood that unless otherwise specified in the present invention, any value between the two endpoints of each numerical range and the two endpoints themselves can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials similar to or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0069] In the following examples and comparative examples of this application, the plastic centrifuge tubes and grinding tubes used in the method for extracting total RNA from cartilage tissue were autoclaved at 121 °C for 30 min and then dried for standby; pipette tips (sterile and enzyme-free, purchased from axygen); the pipette and the experimental operation table were irradiated with ultraviolet light for 30 min and wiped with RNAzap.

[0070] Diethyl pyrocarbonate (DEPC) aqueous solution: DEPC was added to deionized water to make the concentration 0.1 v / v% and obtained by autoclaving at high temperature.

[0071] Cetyltrimethylammonium bromide (CTAB) lysis solution, and its formula is: 200 mM Tris-HCl, 100 mM EDTA, 1.5 M NaCl, 2 wt% CTAB, pH 8.0. All reagents were prepared with DEPC aqueous solution and sterilized for standby.

[0072] The particle size of the cartilage tissue powder is 0.12 μm.

[0073] The Trizol extraction solution is composed of phenol, 8-hydroxyquinoline, and guanidine isothiocyanate. The Trizol extraction solution was purchased from Shanghai Maji Yuhua Biomedical Technology Co., Ltd.

[0074] The enzyme activity of proteinase K is 200 U / g.

[0075] Extraction method of total RNA from rat cartilage tissue in Example 1

[0076] In this example, taking rat cartilage tissue as the object, the total RNA of rat cartilage tissue was extracted by using the method of the present invention, Trizol method and EASYspin Plus Bone Tissue RNA kit respectively, and the concentration and integrity of the obtained total RNA were detected. Specifically as follows:

[0077] Example 1.1: Extract total RNA from rat cartilage tissue by using the extraction method of the present invention, including the following steps:

[0078] 1) Take rat cartilage tissue and grind it into powder under liquid nitrogen. Add 1000 μL of CTAB lysis solution to 100 mg of tissue powder and mix well, then add 20 μL of proteinase K solution and incubate at 55 °C for 15 min. Among them, the volume ratio of CTAB lysis solution to β-mercaptoethanol is 100:2; the proteinase K solution is obtained by dissolving proteinase in buffer solution, the concentration of the proteinase K solution is 100 μg / mL, and the buffer solution is composed of 50 mM Tris-HCl (pH 7.4) and 10 mM CaCl2.

[0079] 2) Centrifuge at 4 °C, 14000 g for 2 min, take the supernatant, add phenol-chloroform with the volume of the supernatant, shake well, and let it stand at room temperature for 3 min. Among them, phenol-chloroform is a mixed solution of water-saturated phenol and chloroform, and the volume ratio of water-saturated phenol to chloroform is 1:1.

[0080] 3) Centrifuge at 4 °C, 14000 g for 15 min, take the supernatant, add 0.8 times the volume of absolute ethanol of the supernatant, and shake gently.

[0081] 4) Centrifuge at 4 °C, 14000 g for 10 min, discard the supernatant, and wash the precipitate with 75% (v / v) ethanol twice.

[0082] 5) Centrifuge the washed precipitate at 4 °C, 14000 g for 10 min, discard the supernatant, air-dry the precipitate at room temperature, then dissolve it fully with DEPC aqueous solution and store it at -80 °C.

[0083] Comparative Example 1.2: Extract rat cartilage tissue RNA by Trizol method, including the following steps:

[0084] 1) Take rat cartilage tissue and grind it into powder under liquid nitrogen. Add 1000 μL of Trizol extraction solution to 100 mg of tissue powder and mix well.

[0085] 2) Centrifuge at 4 °C, 14000 g for 2 min, take the supernatant, add 200 μL of chloroform, shake well, and let it stand at room temperature for 3 min.

[0086] 3) Centrifuge at 4°C, 14,000 g for 15 min, take the supernatant, add an equal volume of absolute ethanol to the supernatant, and gently shake well.

[0087] 4) Centrifuge at 4°C, 14,000 g for 10 min, discard the supernatant, and wash the precipitate twice with 75% ethanol by volume.

[0088] 5) Centrifuge at 4°C, 14,000 g for 10 min, discard the supernatant, air-dry the precipitate at room temperature, then dissolve it thoroughly with DEPC aqueous solution and store at -80°C.

[0089] Comparative Example 1.3: Extract RNA from rat cartilage tissue using the EASYspin Plus Bone Tissue RNA kit, including the following steps:

[0090] The EASYspin Plus Bone Tissue RNA kit used for extraction was purchased from Ailepu Biotechnology (Beijing) Co., Ltd.

[0091] 1) Take 100 mg of rat cartilage tissue, add CLB lysis buffer, vortex vigorously, incubate in a water bath at 65°C for 10 min, and then centrifuge at 4°C, 13,000 rpm for 10 min.

[0092] 2) Take the supernatant and add 0.5 volume of absolute ethanol.

[0093] 4) Transfer to a genomic DNA removal column, centrifuge at 13,000 rpm for 2 min, and discard the waste liquid.

[0094] 5) Add 500 μL of lysis buffer RLT Plus and centrifuge at 13,000 rpm for 30 sec.

[0095] 6) Take the filtrate, add 0.5 volume of absolute ethanol, transfer to an adsorption column, centrifuge at 13,000 rpm for 2 min, and discard the waste liquid.

[0096] 7) Add 700 μL of protein removal solution RW1 and centrifuge at 13,000 rpm for 30 sec, then discard the waste liquid.

[0097] 8) Add 500 μL of wash solution RW, centrifuge at 13,000 rpm for 30 sec, discard the waste liquid, and repeat 2 times; add 30 - 50 μL of DEPC water to elute the nucleic acid.

[0098] Use a Nanodrop ultraviolet-visible spectrophotometer and 1.5% agarose gel electrophoresis to detect the concentration and integrity of the RNA obtained in Example 1, Comparative Example 1, and Comparative Example 1#, and the concentration detection results are shown in Table 1, and the integrity detection results are shown in Figures 1 - 3 .

[0099] Table 1

[0100] Extraction scheme Concentration ng / μL OD260 / OD280 OD260 / OD230 Example 1.1 814.50 2.07 2.08 Comparative Example 1.2 26.30 1.94 1.07 Comparative Example 1.3 176.6 2.11 2.05

[0101] Note: Each extraction method was set with three replicates, and the average value was taken.

[0102] From Table 1, Figures 1 - 3 it can be seen that the method of the present invention can extract the complete RNA of rat cartilage tissue, and the concentration and integrity of the nucleic acid are better than those of the Trizol method and the EASYspin Plus Bone Tissue RNA kit; compared with the traditional Trizol method, the concentration is increased by nearly 30 times; compared with the kit, the concentration is increased by nearly 4 times.

[0103] Extraction method of total RNA from mouse cartilage tissue in Example 2

[0104] In this example, taking mouse cartilage tissue as the object, the total RNA of mouse cartilage tissue was extracted by using the method of the present invention, the Trizol method, and the EASYspin Plus Bone Tissue RNA kit respectively, and the concentration and integrity of the obtained total RNA were detected.

[0105] Example 2.1: The difference from 1.1 in Example 1 is that mouse cartilage tissue was used as the object, and the rest was the same as 1.1 in Example 1.

[0106] Comparative Example 2.2: The difference from 1.2 in Example 1 is that mouse cartilage tissue was used as the object, and the rest was the same as 1.2 in Example 1.

[0107] Comparative Example 2.3: The difference from 1.3 in Example 1 is that mouse cartilage tissue was used as the object, and the rest was the same as 1.3 in Example 1.

[0108] The RNA obtained in Example 2.1, Comparative Example 2.2, and Comparative Example 2.3 was detected for concentration and integrity by using a Nanodrop ultraviolet-visible spectrophotometer and 1.5% agarose gel electrophoresis. The concentration detection results are shown in Table 2, and the integrity detection results are shown in Figures 1 - 3 .

[0109] Table 2

[0110] Extraction scheme Concentration ng / μL OD260 / OD280 OD260 / OD230 Example 2.1 1486.30 2.05 2.11 Comparative Example 2.2 2.80 2.1 0.58 Comparative Example 2.3 153.2 2.1 1.76

[0111] Note: Each extraction method was set with three replicates, and the average value was taken.

[0112] From Table 2, Figures 1 - 3It can be seen that the method of the present invention can extract the complete RNA of mouse cartilage tissue, and the concentration and integrity of the nucleic acid are superior to those of the Trizol method and the EASYspin Plus Bone Tissue RNA kit; compared with the traditional Trizol method, the concentration is increased by nearly 530 times; compared with the kit, the concentration is increased by nearly 8 times.

[0113] Example 3 Method for Extracting Total RNA from Rabbit Cartilage Tissue

[0114] In this example, rabbit cartilage tissue was used as the object, and the total RNA of rabbit cartilage tissue was extracted by using the method of the present invention, the Trizol method and the EASYspin Plus Bone Tissue RNA kit respectively, and the concentration and integrity of the obtained total RNA were detected.

[0115] Example 3.1: The difference from 1.1 in Example 1 is that rabbit cartilage tissue was used as the object, and the rest was the same as 1.1 in Example 1.

[0116] Comparative Example 3.2: The difference from 1.2 in Example 1 is that rabbit cartilage tissue was used as the object, and the rest was the same as 1.2 in Example 1.

[0117] Comparative Example 3.3: The difference from 1.3 in Example 1 is that rabbit cartilage tissue was used as the object, and the rest was the same as 1.3 in Example 1.

[0118] The RNA obtained in Example 3.1, Comparative Example 3.2 and Comparative Example 3.3 was detected for concentration and integrity by using a Nanodrop ultraviolet-visible spectrophotometer and 1.5% agarose gel electrophoresis. The concentration detection results are shown in Table 3, and the integrity detection results are shown in Figures 1 - 3 .

[0119] Table 3

[0120] Extraction scheme Concentration ng / μL OD260 / OD280 OD260 / OD230 Example 3.1 348.70 2.05 2.06 Comparative Example 3.2 7.90 2.15 1.53 Comparative Example 3.3 51.60 2.09 1.94

[0121] Note: Each extraction method was set with three replicates and the average value was taken.

[0122] From Table 3, Figures 1 - 3 it can be seen that the method of the present invention can extract the complete RNA of rabbit cartilage tissue, and the concentration and integrity of the nucleic acid are superior to those of the Trizol method and the EASYspin Plus Bone Tissue RNA kit; compared with the traditional Trizol method, the concentration is increased by nearly 43 times; compared with the kit, the concentration is increased by nearly 6 times.

[0123] Example 4 Method for Extracting Total RNA from Chicken Cartilage Tissue

[0124] In this example, chicken cartilage tissue was used as the object, and the total RNA of chicken cartilage tissue was extracted by using the method of the present invention, Trizol method, and EASYspin Plus Bone Tissue RNA kit respectively, and the concentration and integrity of the obtained total RNA were detected.

[0125] Example 4.1: The difference from 1.1 in Example 1 is that chicken cartilage tissue was used as the object, and the rest was the same as 1.1 in Example 1.

[0126] Comparative Example 4.2: The difference from 1.2 in Example 1 is that chicken cartilage tissue was used as the object, and the rest was the same as 1.2 in Example 1.

[0127] Comparative Example 4.3: The difference from 1.3 in Example 1 is that chicken cartilage tissue was used as the object, and the rest was the same as 1.3 in Example 1.

[0128] The RNA obtained in Example 4, Comparative Example 4.1, and Comparative Example 4.2 was detected for concentration and integrity using a Nanodrop ultraviolet-visible spectrophotometer and 1.5% agarose gel electrophoresis. The concentration detection results are shown in Table 4, and the integrity detection results are shown in Figures 1 - 3 .

[0129] Table 4

[0130] Extraction scheme Concentration ng / μL OD260 / OD280 OD260 / OD230 Example 4.1 753.90 2.07 1.59 Comparative Example 4.2 28.60 2.04 1.82 Comparative Example 4.3 34.10 2.03 1.95

[0131] Note: Each extraction method was set with three replicates, and the results were averaged.

[0132] From Table 4, Figures 1 - 3 it can be seen that the method of the present invention can extract the complete RNA of chicken cartilage tissue, and the concentration and integrity of nucleic acid are better than those of the Trizol method and the EASYspin Plus Bone Tissue RNA kit; compared with the traditional Trizol method, the concentration is increased by nearly 25 times; compared with the kit, the concentration is increased by nearly 21 times.

[0133] Example 5 Extraction Method of Total RNA from Coral Tissue

[0134] In this example, coral tissue was used as the object, and the total RNA of coral tissue was extracted by using the method of the present invention, Trizol method, and EASYspinPlus Bone Tissue RNA kit respectively, and the concentration and integrity of the obtained total RNA were detected.

[0135] Example 5.1: The difference from 1.1 in Example 1 is that coral tissue was used as the object and incubated at 50 °C for 20 min, and the rest was the same as 1.1 in Example 1.

[0136] Comparative Example 5.2: The difference from 1.2 in Example 1 is that the coral tissue is used as the object, and the rest is the same as 1.2 in Example 1.

[0137] Comparative Example 5.3: The difference from 1.3 in Example 1 is that the coral tissue is used as the object, and the rest is the same as 1.3 in Example 1.

[0138] The RNA obtained in Example 5.1, Comparative Example 5.2 and Comparative Example 5.3 was detected for concentration and integrity using a Nanodrop ultraviolet-visible spectrophotometer and 1.5% agarose gel electrophoresis. The concentration detection results are shown in Table 5, and the integrity detection results are shown in Figures 1 - 3 .

[0139] Table 5

[0140] Extraction scheme Concentration ng / μL OD260 / OD280 OD260 / OD230 Example 5.1 667.90 2.13 1.99 Comparative Example 5.2 47.90 2.10 1.86 Comparative Example 5.3 81.50 2.14 1.96

[0141] Note: Each extraction method was set with three replicates, and the quality inspection results were averaged.

[0142] From Table 5, Figures 1 - 3 it can be seen that the method of the present invention can extract complete RNA from coral tissue, and the concentration and integrity of nucleic acids are better than those of the Trizol method and the EASYspin Plus Bone Tissue RNA kit; compared with the traditional Trizol method, the concentration is increased by nearly 13 times; compared with the kit, the concentration is increased by nearly 7 times.

[0143] Example 6 Method for Extracting Total RNA from Shrimp Antenna Tissue

[0144] In this example, taking the shrimp antenna tissue as the object, the method of the present application, the Trizol method and the EASYspin Plus Bone Tissue RNA kit were used to extract the total RNA of the shrimp antenna tissue, and the obtained total RNA was detected for concentration and integrity.

[0145] Example 6.1: The difference from 1.1 in Example 1 is that the shrimp antenna tissue is used as the object and incubated at 60 °C for 10 min, and the rest is the same as 1.1 in Example 1.

[0146] Comparative Example 6.2: The difference from 1.2 in Example 1 is that the shrimp antenna tissue is used as the object, and the rest is the same as 1.2 in Example 1.

[0147] Comparative Example 6.3: The difference from 1.3 in Example 1 is that the shrimp antenna tissue is used as the object, and the rest is the same as 1.3 in Example 1.

[0148] The RNA obtained in Example 6, Comparative Example 6, and Comparative Example 6# was detected for concentration and integrity using a Nanodrop ultraviolet-visible spectrophotometer and 1.5% agarose gel electrophoresis. The concentration detection results are shown in Table 6, and the integrity detection results are shown in Figures 1 - 3 .

[0149] Table 6

[0150] Extraction scheme Concentration ng / μL OD260 / OD280 OD260 / OD230 Example 6.1 690.80 2.07 2.25 Comparative Example 6.2 18.20 2.23 0.38 Comparative Example 6.3 40.60 2.32 0.62

[0151] Note: Each extraction method was set with three replicates, and the results were averaged.

[0152] From Table 6, Figures 1 - 3 it can be seen that the method of the present invention can extract intact RNA from shrimp antenna tissues, and the concentration and integrity of the nucleic acid are superior to those of the Trizol method and the EASYspin Plus Bone Tissue RNA kit; compared with the traditional Trizol method, the concentration is increased by nearly 37 times; compared with the kit, the concentration is increased by nearly 16 times.

[0153] Extraction method of total RNA from crab hepatopancreas tissue in Example 7

[0154] In this example, taking crab hepatopancreas tissue as the object, the method of the present invention was used to extract the total RNA of crab hepatopancreas tissue, and the obtained total RNA was detected for concentration and integrity.

[0155] The difference between this example and 1.1 in Example 1 is that crab hepatopancreas tissue was used as the object and incubated at 60 °C for 10 min, and the others were the same as 1.1 in Example 1.

[0156] The RNA obtained in Example 7 was detected for concentration and integrity using a Nanodrop ultraviolet-visible spectrophotometer and 1.5% agarose gel electrophoresis. The concentration detection results are shown in Table 7, and the integrity detection results are shown in Figure 4 .

[0157] Table 7

[0158] Extraction scheme Concentration ng / μL OD260 / OD280 OD260 / OD230 Example 7 481.8 2 1.69

[0159] Note: Each extraction method was set with three replicates, and the average value was taken.

[0160] From Table 7, Figure 4 it can be seen that the RNA extracted from crab hepatopancreas tissue by the method of the present invention is diffused, indicating that the RNA was degraded during extraction.

[0161] Extraction method of total RNA from iris root tissue in Example 8

[0162] In this example, the iris root tissue was used as the object, and the method of the present invention was adopted to extract the total RNA of the iris root tissue, and the concentration and integrity of the obtained total RNA were detected.

[0163] The difference between this example and 1.1 in Example 1 is that the iris root tissue was used as the object and incubated at 60 °C for 10 min, and the others were the same as 1.1 in Example 1.

[0164] The RNA obtained in Example 8 was detected for concentration and integrity using a Nanodrop ultraviolet-visible spectrophotometer and 1.5% agarose gel electrophoresis. The concentration detection results are shown in Table 8, and the integrity detection results are shown in Figure 4 .

[0165] Table 8

[0166] Extraction scheme Concentration ng / μL OD260 / OD280 OD260 / OD230 Example 8 109 1.58 0.89

[0167] Note: Each extraction method was set with three replicates, and the average value was taken.

[0168] From Table 8, Figure 4 it can be seen that the RNA extracted from the iris root tissue by the method of the present invention has good integrity, but poor purity.

[0169] From Examples 1 to 8, it can be seen that the method for extracting RNA of the present invention has a better extraction effect on cartilage tissue than other tissues and plant tissues.

[0170] In summary, by using the extraction method of the present application, the obtained RNA has integrity, a single electrophoresis band and no trailing, and the RNA has high purity, with OD260 / OD280 reaching 2.05 - 2.13, OD260 / OD230 being 1.59 - 2.25, and the concentration being 348.7 = 814.50 ng / μL; while when using the traditional Trizol method for extraction, OD260 / OD280 is 1.94 - 2.15, OD260 / OD230 is 0.58 - 1.86, and the concentration is 2.80 - 47.9 ng / μL; when using a commercial kit for extraction, OD260 / OD280 reaches 2.03 - 2.14, OD260 / OD230 is 1.94 - 2.05, and the concentration is 34.10 - 176.6 ng / μL. Compared with the traditional Trizol method, the concentration of the RNA extracted by the method of the present application is increased by 13 to 530 times; compared with the commercial kit method, the concentration of the RNA extracted by the method of the present application is increased by 4 to 21 times. The method of the present application solves the pretreatment problem for subsequent molecular experiments, qPCR experiments, NGS sequencing and other related experiments, solves the technical problems, greatly improves the success rate of subsequent experiments, and has industrialization prospects.

[0171] The above embodiments are only illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A method for extracting RNA from cartilage tissue, characterized in that, It includes the following steps: 1) Incubate cartilage tissue powder, CTAB lysis buffer, β-mercaptoethanol and proteinase K. 2) Separate the solid and liquid phases, take the supernatant, and mix it with phenol-chloroform. 3) Separate the solid and liquid phases, take the supernatant, and mix it with ethanol. 4) Separate the solid and liquid phases, take the precipitate, and wash it. 5) Separate the solid and liquid phases, take the precipitate, and mix it with a solvent to obtain the RNA described above. The CTAB lysis buffer consists of the following components: 0.5 - 4 wt% CTAB, 0.5 - 4.5 M NaCl, 50 - 400 mM Tris-HCl, and 50 - 200 mM EDTA; the pH value of the CTAB lysis buffer is 7.0 - 9.

0. The incubation temperature is 40 - 70 °C. The incubation time is 5 - 30 min.

2. The method according to claim 1, characterized in that, It includes at least one of the following technical features: A1) The mass-volume ratio of the cartilage tissue powder to the CTAB lysis buffer is 1 mg : (1 - 20) μL. A2) The ratio of the cartilage tissue powder to proteinase K is 100 mg : (0.0001 - 0.002) U. A3) The volume ratio of the CTAB lysis buffer to β-mercaptoethanol is 100 : (0.2 - 5).

3. The method according to claim 1, wherein In 2), the volume ratio of the phenol-chloroform to the supernatant is (0.5 - 2) :

1. And / or, in 3), the volume ratio of the ethanol to the supernatant is (0.5 - 1.5) :

1. And / or, in 4), the washing is performed using an ethanol aqueous solution.

4. The method according to claim 3, wherein In 2), the phenol-chloroform is a mixture of water-saturated phenol and chloroform. And / or, in 3), the ethanol is anhydrous ethanol. And / or, in 4), the concentration of the ethanol aqueous solution is 50 - 80 v / v%.

5. The method according to claim 4, wherein The volume ratio of the water-saturated phenol to the chloroform is (0.5 - 1.5) :

1.

6. The method according to claim 1, wherein In 4), the solvent is a diethyl pyrocarbonate aqueous solution.

7. The method according to claim 6, wherein The concentration of the diethyl pyrocarbonate aqueous solution is 0.05 - 0.2 v / v%.

8. The method according to claim 1, wherein The solid-liquid separation is centrifugation, and the centrifugation conditions are: 3 - 5 °C, 13000 - 17000 g, centrifugation for 5 - 20 min.

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