RNA (Ribonucleic Acid) preservation tube

Abstract

The present invention relates to the technical field of biological material preservation, and particularly discloses an RNA preservation tube, which comprises: an amino acid component selected from at least one of glycine and a derivative thereof; the EDTA metal chelating agent is selected from at least one of EDTA alkali metal salts and hydrates thereof; a preservative component; optionally comprising a pH indicator; the storage tube is provided with a sealed cavity for storing an RNA solution, and the volume of the cavity is 1-20ml; through combination of a glycine-EDTA synergistic buffer system and an imidazolidinyl urea preservative, long-acting stable preservation of an RNA sample in an extreme environment is realized, metal ions can be dynamically chelated and the pH value is maintained in a range of 6.8-7.5 through a specific ratio of glycine to EDTA salt, a multi-protection mechanism is formed by combining a tube wall nucleic acid adsorption inhibition treatment technology, and the RNA sample can be stably preserved in the extreme environment through the combination of the glycine-EDTA synergistic buffer system and the imidazolidinyl urea preservative. And the loss caused by RNA enzyme activity, acid-base degradation and physical adsorption is effectively blocked, and the product is especially suitable for high-temperature transportation or long-term normal-temperature storage scenes.

Description

Technical Field

[0001] The invention belongs to the technical field of biological material preservation, and particularly relates to an RNA preservation tube. Background Art

[0002] Currently, RNA preservation technology mainly relies on the combination of chemical stabilizers and dedicated preservation tubes to maintain the integrity and functionality of RNA. Similar technologies also involve the use of chelating agents to inhibit RNA enzyme activity by binding to metal ions, or using buffer systems to stabilize the pH value of the solution to reduce the damage to RNA caused by acidic and alkaline environments.

[0003] Although existing technologies have made certain progress, they still have significant limitations. First, existing preservation methods are highly dependent on temperature. For example, although PAXgene tubes support short-term storage at room temperature, long-term storage still requires low temperature, which limits their application in resource-scarce areas or scenarios with insufficient transportation conditions. Second, the protective effect of chemical stabilizers is limited. Especially in complex biological samples, RNA may still be degraded due to residual RNases or oxidative stress. These defects jointly restrict the in-depth application of RNA technology in clinical diagnosis, treatment, agriculture and other fields. Summary of the Invention

[0004] The object of the present invention is to provide an RNA storage tube to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] An RNA preservation tube, comprising:

[0007] An amino acid component selected from at least one of glycine and its derivatives;

[0008] EDTA metal chelating agent, selected from at least one of EDTA alkali metal salts and hydrates thereof;

[0009] preservative ingredients;

[0010] optionally comprising a pH indicator;

[0011] The storage tube has a sealed cavity for storing RNA solution, and the capacity of the cavity is 1-20 ml.

[0012] Preferably, the amino acid component is glycine, and its mass percentage is 3%-15%.

[0013] Preferably, the mass percentage of the EDTA metal chelator is 2%-18%, and is preferably selected from the hydrate form of EDTA potassium salt or sodium salt, including EDTA.2K.2H2O or EDTA.3K.2H2O.

[0014] Preferably, the mass percentage of the preservative component is 1%-15%, and is preferably an imidazolidinyl urea compound.

[0015] Preferably, the pH indicator is a phenolphthalein compound, and the addition amount is 0.0005%-0.5%.

[0016] Preferably, the amino acid component and the EDTA metal chelating agent together form a buffer system to maintain the pH of the solution in the range of 6.0-8.5.

[0017] Preferably, the cavity capacity of the storage tube is 5 ml or 10 ml, and the tube wall thickness is positively correlated with the capacity.

[0018] Preferably, the inner surface of the storage tube is treated to inhibit nucleic acid adsorption, and the material is selected from polypropylene, polyethylene or modified cycloolefin polymer.

[0019] Preferably, it further contains an auxiliary stabilizer, which is selected from at least one of sugars, polyols or surfactants.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The combination of a glycine-EDTA synergistic buffer system and an imidazolidinyl urea preservative achieves long-term stable preservation of RNA samples in extreme environments. The specific ratio of glycine and EDTA salts can dynamically chelate metal ions and maintain the pH in the range of 6.8-7.5. Combined with the tube wall nucleic acid adsorption inhibition treatment technology, a multiple protection mechanism is formed to effectively block the loss caused by RNA enzyme activity, acid-base degradation, and physical adsorption. It is particularly suitable for high-temperature transportation or long-term storage at room temperature.

[0022] (2) The phenolphthalein red indicator provides real-time feedback on the sample status through visual color changes. At the same time, the introduction of auxiliary stabilizers makes the product compatible with storage in a wide temperature range of -20°C to 40°C, reducing the problem of RNA breakage caused by repeated freezing and thawing of traditional cryopreservation tubes. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a process flow chart of the present invention. DETAILED DESCRIPTION

[0024] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] Example 1:

[0026] See also Figure 1 As shown, an RNA preservation tube comprises:

[0027] Glycine 8% (w / w);

[0028] EDTA tripotassium salt dihydrate 10% (w / w);

[0029] Imidazolidinyl urea 5% (w / w);

[0030] Phenolphthalein red 0.01% (w / w);

[0031] Deionized water balance;

[0032] The storage tube has a sealed cavity for storing RNA solution, and the capacity of the cavity is 5 ml.

[0033] The preparation method comprises the following steps:

[0034] Dissolve glycine and EDTA tripotassium salt dihydrate in 60°C deionized water and stir until completely dissolved. Cool to 25°C, add imidazolidinyl urea and phenolphthalein red, and mix thoroughly. Dispense into 5 ml polypropylene tubes (silanized inner wall), with a wall thickness of 1.0 mm. Seal and sterilize (121°C, 15 min).

[0035] Example 2:

[0036] See also Figure 1 As shown, an RNA preservation tube comprises:

[0037] Glycine 5% (w / w);

[0038] EDTA dipotassium salt dihydrate 15% (w / w);

[0039] Imidazolidinyl urea 3% (w / w);

[0040] Phenolphthalein red 0.05% (w / w);

[0041] Trehalose 2% (w / w)* (auxiliary stabilizer);

[0042] Deionized water balance;

[0043] The storage tube has a sealed cavity for storing RNA solution, and the capacity of the cavity is 10 ml.

[0044] The preparation method comprises the following steps:

[0045] A polyethylene tube (with plasma-treated inner wall) with a capacity of 10 ml and a wall thickness of 1.3 mm was used; the remaining steps were the same as in Example 1.

[0046] Application experiment example:

[0047] The experimental group was divided into an experimental group and a control group. The experimental group used RNA preservation tubes (5 ml) prepared according to Example 1, and the control group used commercially available RNA stabilization tubes. The test samples selected were artificially synthesized RNA: 1.5 kb RNA fragment (concentration 1 μg / μl) and biological samples: human liver tissue total RNA (concentration 0.5 μg / μl);

[0048] Artificial RNA and biological samples were mixed at a ratio of 1:1. 200 μl of the mixture was added to the experimental and control tubes (n=3 replicates). The tubes were sealed and placed in a 4°C refrigerator, a 25°C constant temperature box, and a 37°C incubator, respectively. Samples were removed on the 7th, 14th, and 30th day for the following tests:

[0049] RNA integrity: RIN value was determined by Agilent 2100 bioanalyzer (1-10, 10 = complete);

[0050] RNA recovery rate: A260 value was determined by spectrophotometry and calculated as the percentage relative to the initial concentration;

[0051] pH monitoring: by phenolphthalein red color change combined with pH meter calibration (initial pH = 7.2);

[0052] Specific experimental data are shown in Tables 1 and 2 below;

[0053] Table 1: RNA integrity (RIN value) under different storage conditions

[0054] Storage conditions Group 7 days 14 days 30 days 4℃ Experimental group 9.8±0.1 9.5±0.2 9.2±0.3 control group 9.3±0.3 8.1±0.4 6.5±0.5 25℃ Experimental group 9.5±0.2 9.0±0.3 8.6±0.4 control group 8.7±0.4 7.2±0.5 4.8±0.6 37℃ Experimental group 9.0±0.3 8.3±0.4 7.5±0.5 control group 7.5±0.5 5.2±0.7 2.1±0.8

[0055] Table 2: RNA recovery (%) and pH changes.

[0056]

[0057]

[0058] As shown above, the experimental group maintained RIN ≥ 7.5 under the extreme conditions of 30 days / 37°C, which was significantly better than the control group (RIN ≤ 2.1). The RNA recovery rate reached 95.2% at 30 days / 25°C, which was 23.1 percentage points higher than the control group.

[0059] The pH fluctuation range of the experimental group was ≤0.4 (7.2→6.8-7.1), while the pH of the control group decreased by 2.1 units (7.2→5.1). The glycine-EDTA buffer system effectively resisted the acid-base changes caused by temperature.

[0060] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An RNA storage tube, characterized in that: include: An amino acid component selected from at least one of glycine and its derivatives; EDTA metal chelating agent, selected from at least one of EDTA alkali metal salts and hydrates thereof; preservative ingredients; optionally comprising a pH indicator; The storage tube has a sealed cavity for storing RNA solution, and the capacity of the cavity is 1-20 ml.

2. The RNA storage tube according to claim 1, characterized in that: The amino acid component is glycine, and its mass percentage is 3%-15%.

3. The RNA storage tube according to claim 1, characterized in that: The mass percentage of the EDTA metal chelating agent is 2%-18%, and is preferably selected from the hydrate form of EDTA potassium salt or sodium salt, including EDTA.2K.2H2O or EDTA.3K.2H2O.

4. The RNA storage tube according to claim 1, characterized in that: The mass percentage of the preservative component is 1%-15%, and is preferably an imidazolidinyl urea compound.

5. The RNA storage tube according to claim 1, characterized in that: The pH indicator is a phenolphthalein compound, and the addition amount is 0.0005%-0.5%.

6. The RNA storage tube according to claim 1, characterized in that: The amino acid component and the EDTA metal chelating agent together form a buffer system to maintain the pH of the solution within the range of 6.0-8.

5.

7. The RNA storage tube according to claim 1, characterized in that: The cavity capacity of the storage tube is 5 ml or 10 ml, and the tube wall thickness is positively correlated with the capacity.

8. The RNA storage tube according to claim 1, characterized in that: The inner surface of the storage tube is treated to inhibit nucleic acid adsorption, and the material is selected from polypropylene, polyethylene or modified cycloolefin polymer.

9. The RNA storage tube according to claim 1, characterized in that: The invention also contains an auxiliary stabilizer, which is selected from at least one of sugars, polyols or surfactants.

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