RNA preservative for rana rugosa and method for preparing the same

Abstract

This invention relates to the field of medical biodetection, specifically to an RNA preservation solution for the spiny-breasted frog and its preparation method. The solution comprises the following components in parts by weight: 0.1-0.2 parts modified nano-white clay, 22-23 parts biomimetic liposome dispersion, 28-32 parts citrate-phosphate buffer, 4-6 parts ammonium sulfate, etc. This invention constructs a composite preservation system and innovatively transforms spiny-breasted frog processing byproducts into key functional components, achieving an organic combination of preservation solution formulation design and green recycling of raw materials. This effectively maintains the integrity of the sample, while significantly reducing production costs and environmental burden. It also possesses good compatibility for multi-omics analysis and ease of operation, providing a stable and reliable sample preservation solution for resource conservation, genetic research, and functional component discovery for spiny-breasted frogs and other precious aquatic animals, with broad prospects for industrial application.

Description

Technical Field

[0001] This invention relates to the field of medical biological detection, and in particular to an RNA preservation solution for the spiny-breasted frog and its preparation method. Background Technology

[0002] The spiny-breasted frog is an important and distinctive economic frog species in the mountainous areas of southern my country, with high value for breeding and medicinal purposes. High-quality RNA samples are required for molecular biological research on it, such as gene expression analysis, pathogen detection, and genetic resource assessment. However, spiny-breasted frog samples are often collected from wild stream environments, and the RNA in its tissues (especially tissues rich in fat) is easily degraded by endogenous ribonucleases after being removed from the body. Moreover, temperature fluctuations during long-distance transportation will exacerbate this process.

[0003] In existing technologies, common methods include direct freezing and immersion in organic solvents. While direct freezing can effectively inhibit enzyme activity, it requires stringent low-temperature conditions at the field sampling site, and repeated freeze-thaw cycles can easily lead to RNA breakage. Meanwhile, acidic phenol-guanidine isothiocyanate solutions, represented by organic solvents, can rapidly lyse cells and inhibit RNase, but their strong corrosiveness, toxicity, and the subsequent phase separation operation make them unsuitable for in-situ fixation or long-term storage scenarios that require maintaining tissue integrity, and they are also incompatible with subsequent multi-omics analysis. In addition, existing technologies mostly focus on the stability of the single RNA component, neglecting the potential value of simultaneously preserving multi-omics information such as proteins and metabolites in the collection of rare samples.

[0004] Therefore, based on the relevant technologies mentioned above, there is an urgent need to develop an RNA preservation solution for spiny-breasted frogs and its preparation method. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an RNA preservation solution for spiny-breasted frogs and its preparation method, so as to solve the problems of harsh field sampling conditions, high toxicity of preservation reagents, and easy damage to sample integrity in the prior art.

[0006] To achieve the above objectives, the present invention provides an RNA preservation solution for spiny-breasted frogs and a method for preparing the same.

[0007] A RNA preservation solution for the spiny-breasted frog comprises the following components in parts by weight: 0.1-0.2 parts modified nano-white clay, 22-23 parts biomimetic liposome dispersion, 28-32 parts citrate-phosphate buffer, 4-6 parts ammonium sulfate, 0.4-0.6 parts disodium EDTA, 0.2-0.4 parts sodium ascorbate, 8-12 parts sucrose, 8-12 parts poloxamer 407, 0.8-1.2 parts extract powder of peptide fragments from enzymatic hydrolysis byproducts of spiny-breasted frog oil, and 0.0008-0.0012 parts proteinase K inhibitor; The modified nano clay is an aminosilane coupling agent modified nano clay. The biomimetic liposome dispersion was prepared from soybean phosphatidylcholine, refined spiny-breasted frog oil, cholesterol, α-tocopherol and citrate-phosphate buffer. The citrate-phosphate buffer solution has a concentration of 1 mol / L and a pH of 6.9.

[0008] Preferably, the preparation steps of the modified nano-clay are as follows: Step A1: Add activated clay, anhydrous ethanol and zirconium oxide grinding balls into a ball mill, heat to 20-30℃, rotate at 300-400 rpm, stir for 3-5 hours, and after ball milling, let stand for 20-40 minutes to obtain slurry; Step A2: Add 3-aminopropyltriethoxysilane to a mixed solvent of ethanol and deionized water, stir at 200-300 rpm for 10-20 min to obtain a silane solution; Step A3: Add the silane solution to the slurry, heat to 65-75℃, stir at 200-300 rpm for 5-7 hours until the reaction is complete, centrifuge and wash, ultrasonically disperse, and dry to obtain modified clay.

[0009] By using wet ball milling to nano-size activated clay, its specific surface area is significantly increased, enabling it to more efficiently adsorb trace amounts of heavy metal ions, polar impurities, and oxidation products in samples. At the same time, amino functional groups are introduced into the surface of the nano-clay through aminosilane, which can significantly improve its dispersion stability in polar buffer systems and enhance its compatibility with biomolecules such as RNA and proteins, avoiding non-specific adsorption of target molecules. In addition, by using aqueous phase modification at 65-75℃, the damage to the material structure caused by high temperature can be avoided.

[0010] Preferably, the mass ratio of activated clay, anhydrous ethanol, and zirconium oxide grinding balls in step A1 is 1:2.8-3.2:7.8-8.2; The ratio of 3-aminopropyltriethoxysilane to the mixed solvent in step A2 is 0.056-0.058 g: 1 mL; The volume ratio of ethanol to deionized water in step A2 is 9:1; The mass ratio of the silane solution to the slurry in step A3 is 0.16-0.18:1.

[0011] Preferably, the preparation steps of the biomimetic liposome dispersion are as follows: Step B1: Add soybean phosphatidylcholine, refined spiny frog oil, cholesterol and α-tocopherol to anhydrous ethanol, heat to 40-60℃, stir at 150-250 rpm for 15-25 minutes, and after stirring is complete, obtain an organic phase solution. Step B2: Add the organic phase solution to the citrate-phosphate buffer solution, heat to 40-60℃, rotate at 400-600 rpm, emulsify for 50-70 min, after the reaction is complete, cool to 4-10℃, sonicate at 150-250W for 4-6 min, after sonication, let stand and cool for 10-20 min to obtain the biomimetic liposome dispersion.

[0012] By constructing biomimetic lipid nanovesicles with soybean phosphatidylcholine as the backbone, integrating cholesterol, unsaturated fatty acids (frog oil), and antioxidants (α-tocopherol) from the spiny-breasted frog, and using this structure to mimic the cell membrane, the nanovesicles can rapidly interact with the tissue cell membrane after sample immersion, while stabilizing the cell structure and effectively preventing intracellular RNA leakage and degradation due to cell damage in the early stage of sampling, thus achieving "in-situ fixation". In addition, by encapsulating frog oil and vitamin E in a lipid bilayer, they can exert their antioxidant and membrane regulation biological functions, while avoiding their direct oxidation and inactivation in the aqueous phase.

[0013] Preferably, the mass ratio of soybean phosphatidylcholine, refined spiny-breasted frog oil, cholesterol, α-tocopherol and anhydrous ethanol in step B1 is 1:0.35-0.45:0.08-0.12:0.01-0.03:3.8-4.2. The mass ratio of the organic phase solution to the citrate-phosphate buffer solution in step B2 is 0.13-0.14:1.

[0014] Preferably, the preparation steps of the peptide extract powder from the enzymatic hydrolysis byproduct of spiny-breasted frog oil are as follows: Step C1: Place the lower aqueous phase byproduct of the enzymatic hydrolysis of spiny-breasted frog oil into a beaker, heat to 80-90℃ for 10-20 min, then cool to 20-30 min, rotate at 150-250 rpm, add 1.0 mol / L hydrochloric acid solution, adjust the pH to 4-5, stir for 20-40 min, after stirring is complete, centrifuge to obtain the supernatant; Step C2: Add the supernatant to a 10kDa molecular weight cutoff membrane, with a transmembrane pressure of 1.5-2.5 bar and a circulation flow rate of 0.8-1.2 L / min. Then add a 3kDa molecular weight cutoff membrane, with a transmembrane pressure of 1.5-2.5 bar and a circulation flow rate of 0.8-1.2 L / min. Repeat 3-4 times. After filtration, freeze-dry under vacuum to obtain the peptide extract powder of the enzymatic hydrolysis byproduct of spiny-breasted frog oil.

[0015] Through a multi-stage purification process combining heat treatment to inactivate residual enzymes, isoelectric point precipitation, and tangential flow ultrafiltration, oils, large molecular proteins, salts, and small molecular amino acids in the byproducts were effectively removed. The resulting peptide extract powder has high purity and good solubility, while avoiding interference from impurities in subsequent molecular experiments. In addition, the retained 3-10kDa peptides contain natural RNase inhibitory activity fragments, providing the preservation solution with a protective component derived from the sample itself and with excellent biocompatibility, thus realizing the full-chain resource utilization.

[0016] A method for preparing an RNA preservation solution for the spiny-breasted frog, the method being as follows: Step S1: Add ammonium sulfate, disodium ethylenediaminetetraacetate, sodium ascorbate and sucrose to citrate-phosphate buffer, heat to 20-30℃, stir at 250-350 rpm for 35-45 minutes, and after stirring is complete, a mixed solution is obtained. Step S2: Add the biomimetic liposome dispersion to the mixture, heat to 20-30℃, stir at 250-350 rpm for 15-25 min, cool to 2-6℃, add poloxamer 407, reduce the speed to 100-200 rpm, stir for 12-14 h, and after stirring is complete, a transparent solution is obtained. Step S3: Add the peptide extract powder of the enzymatic hydrolysis byproduct of spiny-breasted frog oil and proteinase K inhibitor E-64 to a clear solution, heat to 20-30℃, stir at 150-250 rpm for 50-70 min, add modified nano clay, increase the speed to 8000-10000 rpm, homogenize for 3-5 min, reduce the speed to 150-250 rpm, stir for 8-12 min, and obtain the RNA preservation solution for spiny-breasted frog.

[0017] By following the scientific sequence of "first dissolving small molecule stabilizers, then integrating biomimetic structures, followed by adding active ingredients, and finally dispersing nanomaterials," the full dissolution of salts and antioxidants is ensured. The temperature-sensitive matrix poloxamer 407 is fully hydrated at low temperatures to form a homogeneous solution, and the active peptides and inhibitors are completely dissolved. This results in the uniform dispersion of nano-clay without agglomeration, ensuring that each functional component in the final product can fully exert its effect. At the same time, high-speed homogenization ensures that the nano-modified clay can be uniformly dispersed in viscous solution systems, preventing sedimentation and ensuring that the content of adsorption and purification components in each packaged product is consistent, thereby guaranteeing the uniformity and stability of product quality.

[0018] Preferably, the mass ratio of ammonium sulfate, disodium ethylenediaminetetraacetate, sodium ascorbate, sucrose and citrate-phosphate buffer in step S1 is 1:0.08-0.12:0.05-0.07:1.8-2.2:1.8-2.2.

[0019] Preferably, the mass ratio of the biomimetic liposome dispersion / mixture to poloxamer 407 in step S2 is 0.87-0.89:1:0.37-0.39.

[0020] Preferably, in step S3, the mass ratio of the peptide extract powder of the spiny-breasted frog oil enzymatic hydrolysis byproduct, proteinase K inhibitor E-64, transparent solution and modified nano clay is 0.016-0.018:0.00001-0.00003:1:0.0016-0.0018.

[0021] The beneficial effects of this invention are: This invention provides an RNA preservation solution for spiny-breasted frogs and its preparation method. The invention constructs a composite preservation system integrating biomimetic membrane stabilization, temperature-responsive release, endogenous enzyme inhibition, and impurity adsorption. It innovatively transforms spiny-breasted frog processing byproducts into key functional components, achieving an organic combination of preservation solution formulation design and green raw material recycling. Compared with existing technologies, this preservation solution provides excellent in-situ protection of spiny-breasted frog tissue RNA under non-ideal storage conditions, effectively maintaining its integrity. Simultaneously, this technology significantly reduces production costs and environmental burden, and possesses good compatibility for multi-omics analysis and ease of operation. It provides a stable and reliable sample preservation solution for resource conservation, genetic research, and functional component discovery for spiny-breasted frogs and other precious aquatic animals, and has broad prospects for industrial application. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for this invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a bar chart comparing the RNA concentration of the RNA preservation solution in this invention under different environments. Figure 2 This is a bar chart comparing the RNA purity (A260 / A280) of the RNA preservation solution in this invention under different environments. Figure 3 This is a bar chart comparing the RNA purity (A260 / A230) of the RNA preservation solution in this invention under different environments. Figure 4 This is a bar chart comparing the RNA yield of the RNA preservation solution in this invention under different environments. Figure 5 This is a bar chart comparing the RIN values ​​of the RNA preservation solution in this invention under different environments; Figure 6This is a bar chart comparing the RNA stability test results of the RNA preservation solution in this invention. Figure 7 This is a bar chart comparing the biocompatibility of the RNA preservation solution in this invention. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0025] Example 1: Preparation of a modified nano-clay S1: Add 100g of activated clay, 280g of anhydrous ethanol and 780g of zirconium oxide grinding balls to a ball mill, heat to 20℃, rotate at 400rpm, stir for 3h, after ball milling is completed, let stand for 40min to obtain slurry; S2: Add 5.6g of 3-aminopropyltriethoxysilane to a mixed solvent of 90mL ethanol and 10mL deionized water, stir at 200rpm for 20min to obtain a silane solution. S3: Add 16g of silane solution to 100g of slurry, heat to 65℃, stir at 300rpm for 5h until the reaction is complete, centrifuge and wash, ultrasonically disperse, and dry to obtain modified clay.

[0026] Example 2: Preparation of a modified nano-clay S1: Add 100g of activated clay, 300g of anhydrous ethanol and 800g of zirconium oxide grinding balls to a ball mill, heat to 25℃, rotate at 350rpm, stir for 4h, after ball milling is completed, let stand for 30min to obtain slurry; S2: Add 5.7g of 3-aminopropyltriethoxysilane to a mixed solvent of 90mL ethanol and 10mL deionized water, stir at 250rpm for 15min to obtain a silane solution. S3: Add 17g of silane solution to 100g of slurry, heat to 70℃, stir at 250rpm for 6h until the reaction is complete, centrifuge and wash, ultrasonically disperse, and dry to obtain modified clay.

[0027] Example 3: Preparation of a modified nano-clay S1: Add 100g of activated clay, 320g of anhydrous ethanol and 820g of zirconium oxide grinding balls to a ball mill, heat to 30℃, rotate at 300rpm, stir for 5h, after ball milling is completed, let stand for 20min to obtain slurry; S2: Add 5.8g of 3-aminopropyltriethoxysilane to a mixed solvent of 90mL ethanol and 10mL deionized water, stir at 300rpm for 10min to obtain a silane solution. S3: Add 18g of silane solution to 100g of slurry, heat to 75℃, stir at 200rpm for 7h until the reaction is complete, centrifuge and wash, ultrasonically disperse, and dry to obtain modified clay.

[0028] Example 4: Preparation of a biomimetic liposome dispersion S1: Add 10g of soybean phosphatidylcholine, 3.5g of refined spiny frog oil, 0.8g of cholesterol and 0.1g of α-tocopherol to 38g of anhydrous ethanol, heat to 40℃, stir at 250rpm for 15min, and after stirring is complete, obtain an organic phase solution. S2: Add 13g of organic phase solution to 100g of citrate-phosphate buffer, heat to 40℃, rotate at 600rpm, emulsify for 50min, after the reaction is complete, cool to 10℃, sonicate at 150W for 6min, after sonication is finished, let stand and cool for 10-20min to obtain biomimetic liposome dispersion.

[0029] Example 5: Preparation of a biomimetic liposome dispersion S1: Add 10g of soybean phosphatidylcholine, 4g of refined spiny frog oil, 1g of cholesterol and 0.2g of α-tocopherol to 40g of anhydrous ethanol, heat to 50℃, stir at 200rpm for 20min, and after stirring is complete, an organic phase solution is obtained. S2: Add 13.5g of organic phase solution to 100g of citrate-phosphate buffer, heat to 50℃, rotate at 500rpm, emulsify for 60min, after the reaction is complete, cool to 7℃, sonicate at 200W for 5min, after sonication is finished, let stand and cool for 15min to obtain biomimetic liposome dispersion.

[0030] Example 6: Preparation of a biomimetic liposome dispersion S1: Add 10g of soybean phosphatidylcholine, 4.5g of refined spiny frog oil, 1.2g of cholesterol and 0.3g of α-tocopherol to 42g of anhydrous ethanol, heat to 60℃, stir at 150rpm for 25min, and after stirring is complete, obtain an organic phase solution. S2: Add 14g of organic phase solution to 100g of citrate-phosphate buffer, heat to 40℃, rotate at 600rpm, emulsify for 50min, after the reaction is complete, cool to 10℃, sonicate at 150W for 6min, after sonication is finished, let stand and cool for 10min to obtain biomimetic liposome dispersion.

[0031] Example 7: Preparation of a peptide extract powder from enzymatic hydrolysis byproducts of spiny-breasted frog oil S1: Place 100g of the lower aqueous phase byproduct of the enzymatic hydrolysis of spiny-breasted frog oil into a beaker, heat to 80℃ and hold for 20min, then cool to 20min, rotate at 250rpm, add 1.0mol / L hydrochloric acid solution, adjust the pH to 4-5, stir for 20min, after stirring is complete, centrifuge to obtain the supernatant. S2: Add the supernatant to a 10kDa molecular weight cutoff membrane, with a transmembrane pressure of 1.5 bar and a circulation flow rate of 1.2 L / min. Then add a 3kDa molecular weight cutoff membrane, with a transmembrane pressure of 1.5 bar and a circulation flow rate of 1.2 L / min. Repeat this process 3 times. After filtration, freeze-dry under vacuum to obtain the peptide extract powder of the enzymatic hydrolysis byproduct of spiny-breasted frog oil.

[0032] Example 8: Preparation of a peptide extract powder from enzymatic hydrolysis byproducts of spiny-breasted frog oil S1: Place 100g of the lower aqueous phase byproduct of the enzymatic hydrolysis of spiny-breasted frog oil into a beaker, heat to 85℃ for 15min, then cool to 25min, rotate at 200rpm, add 1.0mol / L hydrochloric acid solution, adjust the pH to 4-5, stir for 30min, after stirring is complete, centrifuge to obtain the supernatant. S2: Add the supernatant to a 10kDa molecular weight cutoff membrane, with a transmembrane pressure of 2 bar and a circulation flow rate of 1 L / min. Then add a 3kDa molecular weight cutoff membrane, with a transmembrane pressure of 2 bar and a circulation flow rate of 1 L / min. Repeat this process 4 times. After filtration, freeze-dry under vacuum to obtain the peptide extract powder of the enzymatic hydrolysis byproduct of spiny-breasted frog oil.

[0033] Example 9: Preparation of a peptide extract powder from enzymatic hydrolysis byproducts of spiny-breasted frog oil S1: Place 100g of the lower aqueous phase byproduct of the enzymatic hydrolysis of spiny-breasted frog oil into a beaker, heat to 90℃ and hold for 10min, then cool to 30min, rotate at 150rpm, add 1.0mol / L hydrochloric acid solution, adjust the pH to 4-5, stir for 40min, after stirring is complete, centrifuge to obtain the supernatant. S2: Add the supernatant to a 10kDa molecular weight cutoff membrane, with a transmembrane pressure of 2.5 bar and a circulation flow rate of 0.8 L / min. Then add a 3kDa molecular weight cutoff membrane, with a transmembrane pressure of 2.5 bar and a circulation flow rate of 0.8 L / min. Repeat this process 4 times. After filtration, freeze-dry under vacuum to obtain the peptide extract powder of the enzymatic hydrolysis byproduct of spiny-breasted frog oil.

[0034] Example 10: A method for preparing an RNA preservation solution for the spiny-breasted frog. S1: Add 100g of ammonium sulfate, 8g of disodium ethylenediaminetetraacetate, 5g of sodium ascorbate and 180g of sucrose to 180g of citrate-phosphate buffer, heat to 20℃, stir at 350rpm for 35min, and after stirring is complete, a mixed solution is obtained. S2: Add 87g of biomimetic liposome dispersion (Example 4) to 100g of mixture, heat to 20°C, stir at 350rpm for 15min, cool to 6°C, add 37g of poloxamer 407, reduce the speed to 100rpm, stir for 14h, and after stirring is complete, a transparent solution is obtained. S3: 1.6g of peptide extract powder from enzymatic hydrolysis byproducts of spiny-breasted frog oil (Example 7) and 1mg of proteinase K inhibitor E-64 were added to 100g of transparent solution. The mixture was heated to 20°C, stirred at 250rpm for 50min, and 0.16g of modified nano clay (Example 1) was added. The stirring speed was increased to 1000rpm and homogenized for 3min. The stirring speed was then reduced to 250rpm and stirred for 8min to obtain RNA preservation solution for spiny-breasted frog.

[0035] Example 11: A method for preparing an RNA preservation solution for the spiny-breasted frog. S1: Add 100g of ammonium sulfate, 10g of disodium ethylenediaminetetraacetate, 6g of sodium ascorbate and 200g of sucrose to 200g of citrate-phosphate buffer solution, heat to 25℃, stir at 300rpm for 40min, and after stirring is complete, a mixed solution is obtained. S2: Add 88g of biomimetic liposome dispersion (Example 5) to 100g of mixture, heat to 25°C, stir at 300rpm for 20min, cool to 4°C, add 38g of poloxamer 407, reduce the speed to 150rpm, stir for 13h, and after stirring is complete, a transparent solution is obtained. S3: 1.7g of peptide extract powder from enzymatic hydrolysis byproducts of spiny-breasted frog oil (Example 8) and 2mg of proteinase K inhibitor E-64 were added to 100g of transparent solution. The mixture was heated to 25°C, stirred at 200rpm for 60min, and 0.17g of modified nano clay (Example 2) was added. The stirring speed was increased to 9000rpm and homogenized for 4min. The stirring speed was then reduced to 200rpm and stirred for 10min to obtain RNA preservation solution for spiny-breasted frog.

[0036] Example 12: A method for preparing an RNA preservation solution for the spiny-breasted frog. S1: Add 100g of ammonium sulfate, 12g of disodium ethylenediaminetetraacetate, 7g of sodium ascorbate and 220g of sucrose to 220g of citrate-phosphate buffer solution, heat to 30℃, stir at 250rpm for 45min, and after stirring is complete, a mixed solution is obtained. S2: Add 89g of biomimetic liposome dispersion (Example 6) to 100g of mixture, heat to 30°C, stir at 250rpm for 25min, cool to 2°C, add 39g of poloxamer 407, reduce the speed to 200rpm, stir for 12h, and after stirring is complete, a transparent solution is obtained. S3: 1.8g of peptide extract powder from enzymatic hydrolysis byproducts of spiny-breasted frog oil (Example 9) and 3mg of proteinase K inhibitor E-64 were added to 100g of transparent solution. The mixture was heated to 30°C, stirred at 150rpm for 70min, and 0.18g of modified nano clay (Example 3) was added. The stirring speed was increased to 8000rpm and homogenized for 5min. The stirring speed was then reduced to 150rpm and stirred for 12min to obtain RNA preservation solution for spiny-breasted frog.

[0037] Example 13: A method for preparing an RNA preservation solution for the spiny-breasted frog. S1: Add 100g of ammonium sulfate, 8g of disodium ethylenediaminetetraacetate, 5g of sodium ascorbate, 180g of sucrose, 5g of guanidine hydrochloride, 0.5g of potassium sorbate, and 0.1g of L-cysteine ​​to 180g of citrate-phosphate buffer solution, heat to 20℃, stir at 350rpm for 35min, and after stirring is complete, a mixed solution is obtained; S2: Add 87g of biomimetic liposome dispersion (Example 4) and 20g of medical grade glycerin to 100g of the mixture, heat to 20°C, stir at 350 rpm for 15 min, cool to 6°C, add 37g of poloxamer 407, reduce the stirring speed to 100 rpm, stir for 14 h, and after stirring is complete, a transparent solution is obtained. S3: 1.6g of peptide extract powder from enzymatic hydrolysis byproducts of spiny-breasted frog oil (Example 7) and 1mg of proteinase K inhibitor E-64 were added to 100g of transparent solution. The mixture was heated to 20°C, stirred at 250rpm for 50min, and 0.16g of modified nano clay (Example 1) was added. The stirring speed was increased to 1000rpm and homogenized for 3min. The stirring speed was then reduced to 250rpm and stirred for 8min to obtain RNA preservation solution for spiny-breasted frog.

[0038] Comparative Example 1: Compared with Example 10, this comparative example did not add biomimetic liposome dispersion in the preparation process of an RNA preservation solution for spiny-breasted frogs. All other steps and parameters were the same, and will not be repeated here. The final RNA preservation solution for spiny-breasted frogs was obtained.

[0039] Comparative Example 2: This comparative example differs from Example 10 only in that "modified nano clay" is replaced with "activated clay". All other steps and parameters are the same, and will not be repeated here. The final RNA preservation solution for spiny-breasted frogs is obtained.

[0040] Comparative Example 3: Compared with Example 10, this comparative example did not add poloxamer 407 in the preparation process of an RNA preservation solution for spiny-breasted frogs. All other steps and parameters were the same, and will not be repeated here. The final RNA preservation solution for spiny-breasted frogs was obtained.

[0041] Comparative Example 4: Compared with Example 10, this comparative example did not add spiny-breasted frog oil enzymatic hydrolysis byproduct peptide extract powder during the preparation of a spiny-breasted frog RNA preservation solution. All other steps and parameters were the same, and will not be repeated here. The final result was a spiny-breasted frog RNA preservation solution.

[0042] Comparative Example 5: This comparative example uses JSK-Clove RNA preservation solution.

[0043] Performance testing: RNA integrity test: (refer to) Figure 1-5 ) The Agilent Bioanalyzer 2100 bioanalyzer was used in accordance with the RNA quality assessment methods and interpretation guidelines. 1. Take 1.0 mL of the preservation solution of Examples 10-13 and Comparative Examples 1-5 respectively, add 50 mg of fresh spiny-breasted frog back muscle, two portions of each, and place them in constant temperature incubators. Group A is set at 4℃ and stored for 7 days. Group B is set at 4℃ and stored for 12 hours, then set at 25℃ and stored for 12 hours, for a total of 7 days. 2. Remove the tissue block from the preservation solution, gently blot off any residual preservation solution on the surface with sterile filter paper, add it to a homogenization tube containing lysis buffer, and homogenize thoroughly using a tissue homogenizer. Elute the RNA with RNase-free water, then elute with 30 μL of RNase-free water. Finally, use NanoDropOne to determine the concentration of the extracted RNA, A260 / A280, and A260 / A230, and record the data. 3. Formula for calculating RNA yield: ; 4. Dilute the extracted RNA with RNase-free water to a concentration of approximately 50-100 ng / μL. Take 1 μL of the diluted RNA sample, analyze it using an analyzer, and record the data.

[0044]

[0045]

[0046] RNA stability test (refer to) Figure 6 ) 1. Solution preparation: Add 40 mL of chloroform to 60 mL of glacial acetic acid and mix well to obtain a chloroform-glacial acetic acid mixture; Add 14.0g of potassium iodide to 10mL of deionized water and heat gently to dissolve, thus obtaining a saturated potassium iodide solution. Add 1.0g of starch to 100mL of deionized water, heat to 100℃, stir to dissolve, and obtain starch indicator solution; Add 0.496 g of sodium thiosulfate to deionized water, stir to dissolve, and make up to 1000 mL to obtain sodium thiosulfate standard solution. 2. Peroxide value determination: Take 5.0 g of the preservation solution from Examples 10-13 and Comparative Examples 1-5 respectively, add 30 mL of chloroform-glacial acetic acid mixture, shake well, add 1.0 mL of saturated potassium iodide solution, shake well for 30 s, let stand in the dark for 3 min, add 100 mL of deionized water, titrate with sodium thiosulfate standard solution until pale yellow, add 1 mL of starch indicator, the solution turns blue, continue titrating until the blue color just disappears, record the volume of sodium thiosulfate standard solution consumed (V, unit: mL). For the blank group, except that no sample is weighed, all other operations are exactly the same, and record the volume of sodium thiosulfate standard solution consumed in the blank group (V0, unit: mL). 3. Calculation:

[0047] In the formula: V: the volume of sodium thiosulfate standard solution consumed by the sample, mL; V0: Volume of sodium thiosulfate standard solution consumed in the blank, mL; C: The accurate concentration of the sodium thiosulfate standard solution, in mol / L; m: Sample mass, g; 1000: Conversion factor; 4. Acid value determination: Take 2.0 g of the preservation solution of Examples 10-13 and Comparative Examples 1-5 respectively, add 50 mL of titration solvent (toluene:isopropanol:water volume ratio of 40:59.5:0.5), add 0.05 mol / L potassium hydroxide isopropanol standard solution, and determine the acid value using an automatic potentiometric titrator until the endpoint potential is reached. Record the volume V of KOH standard solution consumed. At the same time, perform a blank control group experiment and record the volume V0 of KOH standard solution consumed. 5. Calculation:

[0048] Where: V: the volume of KOH standard solution consumed by the sample, mL; V0: Volume of KOH standard solution consumed in the blank, in mL; C: The accurate concentration of the KOH standard solution, in mol / L; m: Sample mass, g; 56.11: Molar mass of potassium hydroxide, g / mol;

[0049] Biocompatibility testing (refer to) Figure 7 ) The test was conducted using an ELISA reader in accordance with the GB / T16886.5-2017 testing standard. 1. Add mouse fibroblasts to the culture medium and culture them at 37°C and 5% CO2 until the logarithmic growth phase. Wash them with PBS buffer, add trypsin, stop digestion with complete culture medium, and mix well to form a single-cell suspension. 2. Adjust the cell suspension to 5×10⁻⁶ with complete culture medium. 4 The cells were placed at a density of cells / mL. 100 μL of cell suspension was added to each well of a 96-well plate, and the plates were incubated for 24 hours. 3. Sample dilution: 100% concentration group: dilute the stock solution with culture medium at a ratio of 1:10 (v / v); 10% concentration group: dilute the stock solution with culture medium at a ratio of 1:100 (v / v). 4. Experimental group: Take 100 μL of the preservation solution of Examples 10-13 and Comparative Examples 1-5 respectively, add them to the sample culture medium respectively, and continue to incubate for 24 h; Negative control group: Add 100 μL of fresh complete culture medium; Positive control group: Add 100 μL of complete culture medium containing 10% DMSO; 5. Add 10 μL CCK-8 solution and continue culturing for 4 hours. Measure the optical density at 450 nm using a microplate reader. 6. Calculation:

[0050] 7. Result Judgment Criteria Cell viability ≥80%: The sample is not cytotoxic.

[0051] Cell viability of 60%-80%: The sample showed mild cytotoxicity.

[0052] Cell viability between 40% and 60% indicates moderate cytotoxicity in the sample.

[0053] Cell viability <40%: The sample has severe cytotoxicity.

[0054]

[0055] Data Analysis: As can be seen from Tables 1-4, the RNA preservation solution for spiny-breasted frogs prepared in this invention has superior RNA integrity protection, chemical stability, and biocompatibility. In contrast, Comparative Example 1, lacking the addition of biomimetic liposome dispersion, showed a significant decrease in RNA yield, purity, and integrity (RIN value). Degradation was exacerbated, especially under fluctuating temperature conditions. The fundamental reason for this is the lack of lipid nanostructures that can mimic cell membranes, which prevents the formation of "in-situ fixation" upon contact with tissues, leading to early damage to cell structures and RNA leakage. Furthermore, the absence of the physical barrier of the lipid bilayer makes it easier for endogenous RNases to contact and degrade RNA. In addition, the lack of an antioxidant microenvironment composed of spiny-breasted frog oil and α-tocopherol weakens the overall resistance of the system to oxidative stress, thus affecting the stable preservation of RNA in multiple ways. Comparative Example 2 used only unmodified activated clay, which resulted in a decrease in RNA extraction purity (A260 / A230) and an increase in system peroxide value. The direct cause was that the unmodified clay had a small specific surface area and a lack of surface functional groups, resulting in a weak adsorption capacity for impurities such as salts and organic acids in the RNA extract. At the same time, it had poor dispersibility and was prone to aggregation in polar buffer systems, causing uneven component distribution. In addition, due to the lack of metal ion chelation and free radical scavenging ability after aminosilane modification, the secondary protection mechanism for antioxidant properties of the system was missing, which together affected the RNA purity and the chemical stability of the preservation solution. Comparative Example 3, without the addition of poloxamer 407, resulted in a significant decrease in RNA integrity (RIN value) and a significant increase in the system's acid value. The core reason for this was the lack of encapsulation and stabilization by the thermosensitive polymer gel network, which led to uneven distribution, easy sedimentation, or inactivation of functional components such as biomimetic liposomes and active peptides in the solution. At the same time, under temperature change conditions, the system could not achieve controlled and sustained release of components, reducing the durability and consistency of the protective effect. In addition, the absence of poloxamer 407 also caused the preservation solution to lose its role as a physical stabilizer in maintaining the overall viscosity and microstructure, thereby accelerating component degradation and rancidity.

[0056] Comparative Example 4, which did not include the extract powder of peptide fragments from the enzymatic hydrolysis byproducts of spiny-breasted frog oil, resulted in a decrease in both RNA yield and RIN value. The main reason for this was the lack of a natural RNase-inhibiting peptide derived from the sample itself, leading to insufficient biospecific inhibition of endogenous RNases. At the same time, the absence of this peptide, a biocompatible active ingredient, also weakened the biocompatibility between the preservation solution and the tissue, affecting the penetration and retention of the protective components. Furthermore, the membrane stabilizing auxiliary function that the peptide might possess could not be exerted, thus reducing the RNA protection effect at both the enzyme inhibition and structural synergistic levels.

[0057] Comparative Example 5 used a commercially available general-purpose RNA preservation solution. Although its various indicators were better than those of the aforementioned comparative examples, they were still significantly lower than those of the embodiments of this invention. The fundamental reason for this is that the commercially available products were designed for broad applicability without being specifically tailored to the tissue characteristics of the spiny-breasted frog. This resulted in a lack of in-situ stabilization ability of the biomimetic lipid membrane for high-lipid tissues. In addition, its formulation did not contain species-derived functional peptides and modified nano-adsorbent materials, making it impossible to achieve biologically specific inhibition and targeted removal of impurities. Furthermore, the commercially available solutions have not been integrated and optimized in terms of temperature-sensitive drug loading and multi-omics compatibility. Therefore, their protective efficacy has a clear upper limit when facing complex samples and fluctuating environments.

[0058] Example 13: The introduction of guanidine hydrochloride and L-cysteine ​​resulted in a triple-strength defense of chemical denaturation, enzyme activity inhibition and antioxidant complexation. At the same time, the addition of potassium sorbate provided antibacterial protection and ensured the activity of the components under field conditions. In addition, the addition of glycerol further stabilized the osmotic pressure and membrane structure at low temperature. Therefore, its RNA integrity, chemical stability and biocompatibility all reached their peak.

[0059] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

[0060] This invention is intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. An RNA preservation solution for the spiny-breasted frog, characterized in that, It is composed of the following components in parts by weight: 0.1-0.2 parts modified nano clay, 22-23 parts biomimetic liposome dispersion, 28-32 parts citrate-phosphate buffer, 4-6 parts ammonium sulfate, 0.4-0.6 parts disodium EDTA, 0.2-0.4 parts sodium ascorbate, 8-12 parts sucrose, 8-12 parts poloxamer 407, 0.8-1.2 parts peptide extract powder of enzymatic hydrolysis by-products of spiny-breasted frog oil, and 0.0008-0.0012 parts proteinase K inhibitor; The modified nano clay is an aminosilane coupling agent modified nano clay. The biomimetic liposome dispersion was prepared from soybean phosphatidylcholine, refined spiny-breasted frog oil, cholesterol, α-tocopherol and citrate-phosphate buffer. The citrate-phosphate buffer solution has a concentration of 1 mol / L and a pH of 6.

9.

2. The RNA preservation solution for spiny-breasted frogs according to claim 1, characterized in that, The preparation steps of the modified nano clay are as follows: Step A1: Add activated clay, anhydrous ethanol and zirconium oxide grinding balls into a ball mill, heat to 20-30℃, rotate at 300-400 rpm, stir for 3-5 hours, and after ball milling, let stand for 20-40 minutes to obtain slurry; Step A2: Add 3-aminopropyltriethoxysilane to a mixed solvent of ethanol and deionized water, stir at 200-300 rpm for 10-20 min to obtain a silane solution; Step A3: Add the silane solution to the slurry, heat to 65-75℃, stir at 200-300 rpm for 5-7 hours until the reaction is complete, centrifuge and wash, ultrasonically disperse, and dry to obtain modified clay.

3. The RNA preservation solution for spiny-breasted frogs according to claim 2, characterized in that, The mass ratio of activated clay, anhydrous ethanol, and zirconium oxide grinding balls in step A1 is 1:2.8-3.2:7.8-8.2; The ratio of 3-aminopropyltriethoxysilane to the mixed solvent in step A2 is 0.056-0.058 g: 1 mL; The volume ratio of ethanol to deionized water in step A2 is 9:1; The mass ratio of the silane solution to the slurry in step A3 is 0.16-0.18:

1.

4. The RNA preservation solution for spiny-breasted frogs according to claim 1, characterized in that, The preparation steps of the biomimetic liposome dispersion are as follows: Step B1: Add soybean phosphatidylcholine, refined spiny frog oil, cholesterol and α-tocopherol to anhydrous ethanol, heat to 40-60℃, stir at 150-250 rpm for 15-25 minutes, and after stirring is complete, obtain an organic phase solution. Step B2: Add the organic phase solution to the citrate-phosphate buffer solution, heat to 40-60℃, rotate at 400-600 rpm, emulsify for 50-70 min, after the reaction is complete, cool to 4-10℃, sonicate at 150-250W for 4-6 min, after sonication, let stand and cool for 10-20 min to obtain the biomimetic liposome dispersion.

5. The RNA preservation solution for spiny-breasted frogs according to claim 4, characterized in that, The mass ratio of soybean phosphatidylcholine, refined spiny-breasted frog oil, cholesterol, α-tocopherol, and anhydrous ethanol in step B1 is 1:0.35-0.45:0.08-0.12:0.01-0.03:3.8-4.

2. The mass ratio of the organic phase solution to the citrate-phosphate buffer solution in step B2 is 0.13-0.14:

1.

6. The RNA preservation solution for spiny-breasted frogs according to claim 1, characterized in that, The preparation steps of the peptide extract powder from the enzymatic hydrolysis byproduct of spiny-breasted frog oil are as follows: Step C1: Place the lower aqueous phase byproduct of the enzymatic hydrolysis of spiny-breasted frog oil into a beaker, heat to 80-90℃ for 10-20 min, then cool to 20-30 min, rotate at 150-250 rpm, add 1.0 mol / L hydrochloric acid solution, adjust the pH to 4-5, stir for 20-40 min, after stirring is complete, centrifuge to obtain the supernatant; Step C2: Add the supernatant to a 10kDa molecular weight cutoff membrane, with a transmembrane pressure of 1.5-2.5 bar and a circulation flow rate of 0.8-1.2 L / min. Then add a 3kDa molecular weight cutoff membrane, with a transmembrane pressure of 1.5-2.5 bar and a circulation flow rate of 0.8-1.2 L / min. Repeat 3-4 times. After filtration, freeze-dry under vacuum to obtain the peptide extract powder of the enzymatic hydrolysis byproduct of spiny-breasted frog oil.

7. A method for preparing an RNA preservation solution for the spiny-breasted frog according to any one of claims 1-6, characterized in that, The preparation method is as follows: Step S1: Add ammonium sulfate, disodium ethylenediaminetetraacetate, sodium ascorbate and sucrose to citrate-phosphate buffer, heat to 20-30℃, stir at 250-350 rpm for 35-45 minutes, and after stirring is complete, a mixed solution is obtained. Step S2: Add the biomimetic liposome dispersion to the mixture, heat to 20-30℃, stir at 250-350 rpm for 15-25 min, cool to 2-6℃, add poloxamer 407, reduce the speed to 100-200 rpm, stir for 12-14 h, and after stirring is complete, a transparent solution is obtained. Step S3: Add the peptide extract powder of the enzymatic hydrolysis byproduct of spiny-breasted frog oil and proteinase K inhibitor E-64 to a clear solution, heat to 20-30℃, stir at 150-250 rpm for 50-70 min, add modified nano clay, increase the speed to 8000-10000 rpm, homogenize for 3-5 min, reduce the speed to 150-250 rpm, stir for 8-12 min, and obtain the RNA preservation solution for spiny-breasted frog.

8. The method for preparing an RNA preservation solution for the spiny-breasted frog according to claim 7, characterized in that, The mass ratio of ammonium sulfate, disodium ethylenediaminetetraacetate, sodium ascorbate, sucrose and citrate-phosphate buffer in step S1 is 1:0.08-0.12:0.05-0.07:1.8-2.2:1.8-2.

2.

9. The method for preparing an RNA preservation solution for the spiny-breasted frog according to claim 7, characterized in that, The mass ratio of the biomimetic liposome dispersion, mixture, and poloxamer 407 in step S2 is 0.87-0.89:1:0.37-0.

39.

10. The method for preparing an RNA preservation solution for the spiny-breasted frog according to claim 7, characterized in that, The mass ratio of the peptide extract powder from the enzymatic hydrolysis byproduct of the spiny-breasted frog oil, the proteinase K inhibitor E-64, the transparent solution, and the modified nano clay in step S3 is 0.016-0.018:0.00001-0.00003:1:0.0016-0.0018.

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