Efficient extraction method of mineralized tissue RNA applicable across animal categories

Abstract

The invention belongs to the technical field of biology, and particularly relates to an efficient extraction method of mineralized tissue RNA applicable across animal categories. The invention provides a solution combination suitable for mineralized tissue pretreatment, which not only is beneficial to promoting the release of cells from a mineralized matrix, but also can inhibit the activity of ribonuclease to a certain extent, and is beneficial to extracting high-quality RNA (Ribonucleic Acid). Furthermore, the invention also provides an efficient extraction method of mineralized tissue RNA suitable for cross-animal categories, the sample is pretreated by using a solution combination, and then RNA extraction, precipitation and other treatments are sequentially performed, so that the RNA extraction efficiency of the exoskeleton sample is remarkably improved, and the method has good universality and is suitable for large-scale popularization and application. The method is suitable for extracting RNA in cross-animal mineralized tissues, has important biological significance, and lays a methodological foundation for subsequent development of transcriptomics research of exoskeleton dynamic development, analysis of an environmental adaptation mechanism and construction of a biomineralization process regulation and control network.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for efficiently extracting RNA from mineralized tissues applicable across animal phyla. Background Art

[0002] The skeleton of vertebrates is an endoskeleton, primarily composed of hydroxyapatite and collagen. It is widespread throughout the body, possesses a complex cellular structure (e.g., osteoblasts, osteocytes, and osteoclasts), and can continuously reshape and repair itself under hormonal regulation. In contrast, the shells of invertebrates are mostly extracorporeal structures composed of calcium carbonate or chitin. Traditionally, they were thought to lack cellular involvement, primarily forming through exocrine processes. Their mineralized tissues are often viewed as static, inert, and inert, often referred to as "stone."

[0003] This research team has, for the first time, identified a population of potentially functional living cells within the mineralized shell of an invertebrate. Based on this, they successfully extracted total RNA from the mineralized shell for the first time. This not only challenges the conventional assumption that mineralized tissues are "acellular," but also lays the foundation for further investigation into the formation of invertebrate skeletons and their evolutionary relationship with vertebrate bone tissue.

[0004] As a crucial carrier of genetic information, RNA plays a central role in gene expression regulation. Its high-quality extraction is a crucial foundation for molecular biology research. Currently, RNA extraction techniques are divided into two main categories: chemical lysis-organic phase separation (such as the classic TRIzol method) and silica-membrane column purification (such as various commercial kits). During sample pretreatment, for mammalian cells or soft tissue samples, researchers typically use methods such as liquid nitrogen grinding, mechanical homogenization, or proteinase K digestion to achieve complete cell lysis and release of RNA. For cultured cells, the cell and nuclear membranes can be dissolved by directly adding lysis buffer. These established methods are highly applicable in studies of vertebrate tissues (such as liver and muscle) or cell lines.

[0005] However, although existing RNA extraction technologies are quite mature, these conventional methods have obvious limitations when it comes to extracting RNA from invertebrate shells: first, the chitin and calcified matrix rich in invertebrate exoskeletons form a dense barrier, making it difficult for conventional lysis solutions to effectively penetrate, resulting in incomplete release of cellular contents; second, although ultrasonic grinding is a commonly used tissue lysis method, when applied to samples of mineralized tissue, long-term ultrasonic treatment can easily cause RNA breakage and severe fragmentation; in addition, the polysaccharide-protein complexes commonly found in arthropod shells / exoskeletons will form insoluble colloidal precipitates in the TRIzol method, interfering with the phase separation process, and will clog the silica membrane in the column method, significantly reducing RNA binding efficiency. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for efficiently extracting RNA from mineralized tissues that is applicable across animal phyla, has simple operation, high extraction efficiency, good RNA stability and high quality.

[0007] The present invention provides a solution combination suitable for pretreatment of mineralized tissue, the solution combination comprising: a mineralized tissue treatment solution and a mineralized tissue disruption solution; The mineralized tissue treatment solution uses PBS buffer as a solvent, including: 8 w / v%-10 w / v% sodium lauryl sulfate and 70 v / v%-75 v / v% anhydrous ethanol; The mineralized tissue disruption solution uses a mixed solution as a solvent, comprising: 1-1.5 mM carboxyacetic acid chelating agent; The carboxylic acid chelating agent is composed of EDTA, DTPA and Citric-Acid in a mass ratio of 8: (1-1.5): (1-1.5); The mixed solution is composed of β-mercaptoethanol, chloroform isoamyl alcohol and Solution D in a volume ratio of 8-10 μL:150-200 μL:1 mL; The solution D uses DEPC·H2O as a solvent and includes 0.472-0.475 g / mL of guanidine thiocyanate and 0.005-0.006 g / mL of sodium lauroyl anhydride.

[0008] The present invention provides an application of the solution combination described in the above technical solution in improving the efficiency of RNA extraction from mineralized tissue.

[0009] Preferably, the mineralized tissue originates from one or more of: mollusks, brachiopods and arthropods.

[0010] The present invention provides a method for efficiently extracting RNA from mineralized tissues that is applicable across animal phyla, using the solution combination described in the above technical solution, and comprising the following steps: (1) Soaking the mineralized tissue in a mineralized tissue treatment solution, followed by rinsing and cryogenic grinding to obtain a powder sample; (2) mixing the powder sample with the mineralized tissue disruption solution, followed by freezing reaction and centrifugation to obtain a supernatant; (3) performing a first RNA extraction on the supernatant to obtain a first extract; (4) mixing the first extract with a precipitate to perform a first precipitation to obtain a first precipitate; (5) washing the first precipitate for the first time, dissolving the precipitate for the first time, and mixing it with a DNA digestion solution to obtain a mixed solution; (6) subjecting the mixed solution to a second RNA extraction to obtain a second extract; (7) mixing the second extract with the precipitate to perform a second RNA precipitation to obtain a second precipitate; (8) The second precipitate is washed a second time and dissolved a second time to obtain an RNA sample.

[0011] Preferably, the precipitating solution in step (4) comprises: isopropyl alcohol and sodium acetate in a volume ratio of 10:1-1.5; The volume ratio of the first extract to the precipitate is 1:1.

[0012] Preferably, the DNA digestion solution in step (5) uses DEPC water as a solvent and includes: 4v / v%-5v / v% RNase, 20v / v%-25v / v% DNase 10× Buffer, and 26v / v%-30v / v% DNase I.

[0013] Preferably, the mass volume ratio of the powder sample to the mineralized tissue disruption liquid in step (2) is 0.5-0.8 g:1 mL.

[0014] Preferably, the particle size of the powder sample is 90-110 μm.

[0015] Preferably, the reagents used for the first RNA extraction include: chloroform, isoamyl alcohol and water-saturated phenol; The reagents used for the second RNA extraction include: chloroform, isoamyl alcohol and water-saturated phenol; The chloroform isoamyl alcohol is prepared by mixing chloroform and isoamyl alcohol in a volume ratio of 24:1.

[0016] Preferably, during the first RNA extraction, the volume ratio of chloroform / isoamyl alcohol, water-saturated phenol and supernatant is 60:12:35-50.

[0017] Beneficial effects: The present invention provides a solution combination suitable for pretreatment of mineralized tissue, comprising a mineralized tissue treatment solution and a mineralized tissue disruption solution. This solution, by thoroughly cleaning the sample with the mineralized tissue treatment solution and then using a mineralized tissue disruption solution supplemented with a carboxylic acid chelating agent, not only facilitates the release of cells from the mineralized matrix but also inhibits ribonuclease activity to a certain extent. Therefore, the technical solution of the present invention facilitates the successful extraction of high-quality RNA from fresh shell samples, providing direct molecular evidence for the presence of cellular-derived RNA in shell tissue.

[0018] Based on the above technical advantages, the present invention also provides an efficient method for extracting RNA from mineralized tissues that is applicable across animal classes. It is an RNA extraction method for invertebrate exoskeletons. This method not only significantly improves the RNA extraction efficiency of exoskeleton samples, but also has good versatility, achieving the first stable acquisition of high-quality RNA from highly mineralized tissues.

[0019] The technical solution of the present invention has important biological significance: in the study of marine invertebrates, it makes it possible to systematically analyze the molecular mechanisms of exoskeleton formation and environmental response; in the field of evolutionary biology, it provides reliable technical support for comparing the expression differences of biomineralization-related genes in different species; in environmental science, it makes the screening of environmental stress markers based on exoskeleton RNA more accurate and reliable; the high reproducibility and wide applicability of this method not only solve the technical bottlenecks that have long restricted the study of exoskeleton molecular biology, but also lays a methodological foundation for the subsequent transcriptomic research on the dynamic development of the exoskeleton, the analysis of environmental adaptation mechanisms, and the construction of a regulatory network for the biomineralization process. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 Agarose gel electrophoresis images of different fresh scallop shell RNAs provided by the present invention; Figure 2 Schematic diagram of shell partitioning and agarose gel electrophoresis of shell RNA in different regions provided by the present invention; Figure 3 The peak detection graph of the high-throughput sequencing library of the scallop shell RNA provided by the present invention; Figure 4 Agarose gel electrophoresis diagram of different sea pea shell RNAs provided by the present invention; Figure 5 The peak detection graph of the high-throughput sequencing library of the sea bean shell RNA provided by the present invention; Figure 6 Agarose gel electrophoresis diagram of RNA extracted from different centipede shells provided by the present invention; Figure 7 This is a peak map of the centipede shell RNA high-throughput sequencing library provided by the present invention. DETAILED DESCRIPTION

[0022] Unless otherwise specified, the experimental methods used in the present invention are all conventional methods; the materials, reagents, etc. used in the present invention can be obtained from commercial channels.

[0023] In the present invention, when the mineralized tissue is soaked in the mineralized tissue treatment solution, there is no special requirement for the amount of the mineralized tissue treatment solution used, as long as the mineralized tissue is completely submerged.

[0024] In order to further illustrate the present invention, the method provided by the present invention is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0025] The steps for preparing the reagents required in the examples are as follows: The mineralized tissue treatment solution uses PBS buffer as the solvent, containing 10 w / v% sodium dodecyl sulfate and 70 v / v% anhydrous ethanol. That is, 10 g of SDS (sodium dodecyl sulfate) is added to 20 mL of PBS buffer. After complete dissolution, 70 mL of anhydrous ethanol is added, and then PBS buffer is added to make up the volume to 100 mL. Weigh 47.2 g of guanidine thiocyanate and 0.5 g of sodium lauroyl anhydride, and dilute to 100 mL with DEPC·H2O to obtain Solution D. EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid) and Citric-Acid (citric acid) are mixed uniformly in a mass ratio of 8:1:1 to obtain a carboxylic acid chelating agent; Mineralized tissue disruption solution: 8 μL of β-mercaptoethanol, 200 μL of chloroform isoamyl alcohol, 1 mL of Solution D, and 1 mM carboxyacetic acid chelating agent were mixed evenly to obtain; Precipitation solution: Mix -20°C precooled isopropanol and 3 M sodium acetate in a volume ratio of 10:1; DNA digestion solution: 2 μL RNase, 10 μL DNase 10× Buffer, 13 μL DNase I, and 25 μL DEPC water; Chloroform isoamyl alcohol (or 24:1 chloroform isoamyl alcohol) is a mixture of chloroform and isoamyl alcohol in a volume ratio of 24:1.

[0026] Example 1 Research object - Representative animal of mollusc group, the scallop ( Mizuhopecten yessoensis ) A highly efficient method for extracting RNA from mineralized tissues applicable across animal phyla, comprising the following steps: (1) One-year-old scallops were taken, the soft tissue in the shells was removed, and the shells were completely immersed in a mineralized tissue treatment solution for 2 minutes to achieve rapid shell processing. The shells were then carefully rinsed with deionized water to further clean the shell surface to obtain shells without soft tissue. After the shell surface was wiped dry, the shells were placed in a low-temperature environment of -150°C for quick freezing to obtain the processed scallop shells. (2) Take about 4-5 g of scallop shells processed in step (1), crush them, and grind them using a pre-cooled medium-throughput tissue grinder at a speed of 1000 r / min for 1-2 min. Prepare a powder sample at -80°C. The particle size of the powder sample is 100 μm. (3) Take 0.5 g of powder sample and place it in a 2 mL centrifuge tube, filling it to the 1 mL mark; add mineralized tissue disruption solution at a mass volume ratio of 0.5 g:1 mL of powder to mineralized tissue disruption solution, mix the sample thoroughly and place it on an ice box (make sure to react in a 0 °C environment), wait for the sample to react with the mineralized tissue disruption solution for 10 minutes (shake the sample evenly once every 2 minutes), and then centrifuge at 12000 rpm and 4 °C for 10 minutes, and take the supernatant (the volume of the supernatant is 350-500 μL at this time); (4) Add 600 μL of 24:1 chloroform-isoamyl alcohol to the supernatant of step (3), then add 120 μL of water-saturated phenol, vortex for 30-60 seconds, centrifuge (12000 rpm, 10 min, 4°C), and take the supernatant to a new centrifuge tube; add 350 μL of 24:1 chloroform-isoamyl alcohol and 350 μL of water-saturated phenol to the new centrifuge tube again, vortex for 30-60 seconds, centrifuge (12000 rpm, 10 min, 4°C), repeat this step until the protein layer is removed, centrifuge for the last time, take the supernatant, and transfer it to a new centrifuge tube (the supernatant volume at this time is 300-450 μL); (5) Add an equal volume of precipitate to the centrifuge tube containing the supernatant; mix thoroughly by inversion and let it settle at -20°C for 60 min, then centrifuge (2000 rpm, 40 min, 4°C); (6) Remove the supernatant and add 1 mL of 75% ethanol pre-cooled at -20°C to wash the precipitate twice. Centrifuge after each wash (13,000 rpm, 10 min, 4°C). After the ethanol is completely evaporated, add 50 μL of RNase & DNase-free H2O to dissolve the precipitate and obtain a resuspension. (7) Add 50 μL of DNA digestion solution to the resuspended solution, then place in a metal bath at 37°C for 60 min to remove DNA and obtain the digested solution; (8) Add 610 μL of DEPC water to each tube of digested solution; then add 300 μL of 24:1 chloroform-isoamyl alcohol and 300 μL of water-saturated phenol; then centrifuge at 4°C and 13,000 rpm for 10 min to obtain the supernatant; Prepare a new EP tube, add 600 μL of 24:1 chloroform-isoamyl alcohol, transfer the supernatant to the new EP tube, vortex for 1 min; centrifuge at 4°C, 13,000 rpm for 10 min, and obtain the supernatant; (9) Transfer the supernatant into a new 1.5 mL EP tube and add the precipitate (the volume ratio of the precipitate to the supernatant is 1:1); vortex for 40 seconds; invert and mix thoroughly, then let it settle at -20°C for 60 minutes to obtain the precipitated sample; (10) Place the precipitated sample in a large centrifuge at 4°C and 13,000 rpm for 30 min; discard the supernatant and add 1 mL of 75% ethanol pre-cooled at -20°C to wash twice; after each wash, centrifuge at 4°C and 13,000 rpm for 10 min to obtain the precipitate; (11) Add 15 μL of DEPC water to the precipitate to dissolve it, obtain RNA, and store it at -80°C.

[0027] (12) The integrity of RNA was checked by agarose gel electrophoresis (1.5%).

[0028] In Example 1, four one-year-old scallops were taken and each was processed according to steps (1) to (12) in Example 1. The results are shown in FIG. Figure 1 (exist Figure 1 Lane 1 is MARKER; lanes 2-5 represent the agarose gel electrophoresis patterns of RNA from four different fresh scallop shells, respectively).

[0029] Combine Figure 1 It can be seen that after RNA is extracted using the technical solution provided by the present invention, although some samples are slightly degraded, two main bands of RNA can still be observed in the agarose gel. It can be seen that the present invention can obtain RNA with a higher content.

[0030] Example 2 Research object - Representative animal of mollusc group, the scallop ( Mizuhopecten yessoensis ) A highly efficient method for extracting RNA from mineralized tissues applicable across animal phyla, comprising the following steps: (1) taking a 1-year-old scallop and a 5-year-old scallop, and treating them respectively according to the treatment method of step (1) of Example 1 to obtain treated scallop shells; (2) Take the processed scallop shells respectively, and Figure 2The shells were separated and marked in different partitions of Figure a. Four samples to be extracted were obtained from the 1-year-old scallop, namely the shell edge area (SE), the middle area of ​​the shell (excluding the flesh column area, SC), the shell flesh column area (S-MS) and the hinge area (SH); four samples to be extracted were obtained from the 5-year-old scallop, namely the shell edge area (SE), the middle area of ​​the shell (excluding the flesh column area, SC), the shell flesh column area (S-MS) and the hinge area (SH); the above eight samples to be extracted were processed according to steps (3) to (12) in Example 1, respectively, to obtain RNA from different areas of the scallop shell; The muscle tissue of the one-year-old Yezo scallop was removed, cleaned, and cut into small pieces, and processed according to steps (3) to (12) in Example 1 to obtain scallop tissue RNA; The RNA samples obtained above were subjected to agarose gel electrophoresis. Figure 2 Figure b (in Figure 2 In Figure b, lane 1 is MARKER; lane 2 is the RNA electrophoresis of scallop tissue; lanes 3-6 are the RNA electrophoresis of the SE, SC, S-MS and SH regions of the 1-year-old scallop shell; lanes 7-10 are the domain RNA electrophoresis of the SE, SC, S-MS and SH regions of the 5-year-old scallop shell). The sample in lane 3 was then prepared using the Novogene RNA library preparation kit, and the library quality was tested using Aginent 2100. The library was qualified, and the library test peak diagram is shown in the figure below. Figure 3 shown.

[0031] Combine Figure 2 、 Figure 3 It can be seen that by using the technical solution provided by the present invention to extract RNA from different parts of the shell, a high content of RNA can be obtained; and according to the results, it can be concluded that RNA is commonly present in different areas of the scallop shell.

[0032] Example 3 Research object - representative animal of brachiopod group, sea bean sprout ( Lingula ) (1) Take three sea pea shells that have been frozen immediately after removing the soft tissue from the shells, take them out of the -80°C freezer, and then quickly freeze them in liquid nitrogen; (2) After the liquid nitrogen is completely frozen, the sea bean shells are crushed into small pieces and ground using a pre-cooled medium-throughput tissue grinder at a speed of 1000 r / min for 1-2 min. The sea bean shells are made into a powder sample at an operating temperature below -80°C (the particle size of the powder sample is 100 μm); The powder samples were then processed according to steps (3) to (12) in Example 1, and the three RNA samples obtained were subjected to agarose gel electrophoresis. The results are shown in Table 1. Figure 4 (exist Figure 4 In the figure, lane a is the shell of sea bean sprout; lane 1 in figure b is MARKER; lanes 2-4 in figure b respectively represent the electrophoresis diagrams of RNA extracted from three sea bean sprout shells); the sample in lane 2 was then used with the Novogene RNA library preparation kit, and the library quality was tested using Aginent 2100. The library was qualified, and the library test peak diagram is shown as follows Figure 5 shown.

[0033] Combine Figure 4 、 Figure 5 It can be seen that by adopting the technical solution provided by the present invention to extract RNA, a high content of RNA can be obtained.

[0034] Example 4 Research object - centipede, a representative animal of the arthropod group ( Scolopendra ) (1) Take 4 centipede shells with the soft tissue removed, take them out of the -80℃ freezer, and then quickly freeze them in liquid nitrogen; (2) After the liquid nitrogen is completely frozen, the centipede shell is crushed into small pieces and ground using a pre-cooled medium-throughput tissue grinder at a speed of 1000 r / min for 1-2 min. The shell is made into a powder sample at an operating temperature below -80°C (the particle size of the powder sample is about 100 μm); The powder samples were then processed according to steps (3) to (12) in Example 1, and the four RNA samples obtained were subjected to agarose gel electrophoresis. The results are shown in Table 1. Figure 6 (exist Figure 6 In the figure, Figure a shows the shell of the centipede; lanes 1-4 in Figure b represent the electrophoresis diagrams of RNA extracted from four samples, and lane 5 in Figure b represents MARKER); the sample in lane 1 was then used with the Novogene RNA library preparation kit, and the library quality was tested using Aginent 2100. The library was qualified, and the library test peak diagram is shown as follows: Figure 7 shown.

[0035] Combine Figure 6 、 Figure 7 It can be seen that by adopting the technical solution provided by the present invention to extract RNA, a high content of RNA can be obtained.

[0036] Example 5 Research object - the effect of powders with different particle sizes on RNA extraction results (1) Three one-year-old scallops were taken, the soft tissue in the shells was removed, and the shells were completely immersed in mineralized tissue treatment solution for 2 minutes to achieve rapid shell processing. The shells were then carefully rinsed with deionized water to further clean the shell surface to obtain shells without soft tissue. After the water on the shell surface was wiped dry, the shells were placed in a low-temperature environment of -150 °C for quick freezing to obtain the processed scallop shells. (2) Take about 2 g of the scallop shells processed in step (1), crush them, and grind them using a pre-cooled medium-throughput tissue grinder at a speed of 1000 r / min for 1-2 min. Make the shells into powder samples at -80°C, observe the crushing of the sample in the middle, take out a part when the powder size is about 500 μm, take out another part when the size is about 100 μm, and take out all the powder when the powder size is less than 90 μm; That is, each sample will produce three powder samples with different particle sizes. The powder samples are processed according to steps (3) to (12) in Example 1, and the RNA concentration of the obtained RNA samples is tested. The results are shown in Table 1.

[0037] Table 1 RNA concentration results of powders with different particle sizes

[0038] Combining the data in Table 1, we can see that when the powder particle diameter is too large (≈500μm) or too small (<90μm), the amount of RNA extracted is lower than when the powder particle diameter is 100μm. Therefore, a powder size of 100μm is most suitable for extracting RNA from mineralized tissue.

[0039] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A solution combination suitable for pretreatment of mineralized tissue, characterized in that: The solution combination comprises: a mineralized tissue treatment solution and a mineralized tissue disruption solution; The mineralized tissue treatment solution uses PBS buffer as a solvent, including: 8 w / v%-10 w / v% sodium lauryl sulfate and 70 v / v%-75 v / v% anhydrous ethanol; The mineralized tissue disruption solution uses a mixed solution as a solvent, comprising: 1-1.5 mM carboxyacetic acid chelating agent; The carboxylic acid chelating agent is composed of EDTA, DTPA and Citric-Acid in a mass ratio of 8: (1-1.5): (1-1.5); The mixed solution consists of β-mercaptoethanol, chloroform isoamyl alcohol and Solution D in a volume ratio of 8-10 μL:150-200 μL:1 mL; The solution D uses DEPC·H2O as a solvent and includes 0.472-0.475 g / mL of guanidine thiocyanate and 0.005-0.006 g / mL of sodium lauroyl anhydride.

2. Use of the solution combination according to claim 1 in improving the efficiency of RNA extraction from mineralized tissue.

3. The use according to claim 2, characterized in that The mineralized tissues may originate from one or more of molluscs, brachiopods and arthropods.

4. A method for efficiently extracting RNA from mineralized tissues applicable across animal phyla, characterized in that: The method is carried out using the solution combination according to claim 1, comprising the following steps: (1) Soaking the mineralized tissue in a mineralized tissue treatment solution, followed by rinsing and cryogenic grinding to obtain a powder sample; (2) mixing the powder sample with the mineralized tissue disruption solution, followed by freezing reaction and centrifugation to obtain a supernatant; (3) performing a first RNA extraction on the supernatant to obtain a first extract; (4) mixing the first extract with the precipitate to perform a first precipitation to obtain a first precipitate; (5) washing the first precipitate for the first time, dissolving the precipitate for the first time, and mixing it with a DNA digestion solution to obtain a mixed solution; (6) subjecting the mixed solution to a second RNA extraction to obtain a second extract; (7) mixing the second extract with the precipitate to perform a second RNA precipitation to obtain a second precipitate; (8) The second precipitate is washed a second time and dissolved a second time to obtain an RNA sample.

5. The efficient extraction method according to claim 4, wherein The precipitating solution in step (4) comprises: isopropyl alcohol and sodium acetate in a volume ratio of 10:1-1.5; The volume ratio of the first extract to the precipitate is 1:

1.

6. The method according to claim 4, characterized in that The DNA digestion solution in step (5) uses DEPC water as a solvent and includes: 4v / v%-5v / v% RNase, 20v / v%-25v / v% DNase 10× Buffer, and 26v / v%-30v / v% DNase I.

7. The efficient extraction method according to claim 4, characterized in that The mass volume ratio of the powder sample to the mineralized tissue fragmentation liquid in step (2) is 0.5-0.8 g:1 mL.

8. The efficient extraction method according to claim 4 or 7, characterized in that The particle size of the powder sample is 90-110 μm.

9. The efficient extraction method according to claim 4, characterized in that The reagents used for the first RNA extraction include: chloroform, isoamyl alcohol and water-saturated phenol; The reagents used for the second RNA extraction include: chloroform, isoamyl alcohol and water-saturated phenol; The chloroform isoamyl alcohol is prepared by mixing chloroform and isoamyl alcohol in a volume ratio of 24:

1.

10. The efficient extraction method according to claim 4 or 9, characterized in that: During the first RNA extraction, the volume ratio of chloroform / isoamyl alcohol, water-saturated phenol and supernatant was 60:12:35-50.

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