RNA preservation solution and use thereof

Abstract

Provided is an RNA preservation solution and the use thereof. The RNA preservation solution comprises a protein denaturant, a chelating agent, a reducing agent and a pH buffer, and the pH of the preservation solution is 3.5-5.0. The protein denaturant is used for inhibiting the activity of RNase, and the protein denaturant comprises guanidine isothiocyanate and lithium chloride (LiCl). Compared with known RNA preservation solutions, the preservation solution, after being preserved for a relatively long period of time at different temperatures, can enable RNA to be stably amplified and maintain a relatively high Ct value.

Description

RNA preservation solution and use thereof

[0001] Cross-references

[0002] This invention claims priority from an application filed in China with application number 2023110340757, filed on August 17, 2023, entitled "A RNA Preservation Solution and Uses Thereof." The aforementioned application is incorporated herein by reference in its entirety as a part thereof. Technical Field

[0003] The present invention belongs to the field of medical biological detection, and in particular relates to an RNA preservation solution and application thereof. Background Art

[0004] Ribonucleotides (RNA), ubiquitous in animals, plants, microorganisms, and certain viruses and bacteriophages, serve as carriers of genetic information. While high-quality RNA can be obtained by immediate extraction from fresh peripheral blood or other tissue samples, in practice, immediate extraction from peripheral blood samples is often not possible. Therefore, preserving samples to obtain high-quality RNA is crucial. Commonly used methods for preserving blood samples prior to RNA extraction include cryopreservation, storage in PAX gene whole blood RNA tubes, and the addition of RNA preservation solution to the sample.

[0005] Cryopreservation is currently a common method for processing blood samples. Peripheral blood is collected and stored using ethylenediaminetetraacetic acid (EDTA) anticoagulant tubes. The process is simple, requires no additional processing, and avoids the influence of exogenous factors. However, RNA in peripheral blood is easily affected by endogenous factors, such as the presence of a large amount of ribonuclease (RNase), and RNA is prone to degradation during collection and storage.

[0006] Using PAXgene whole blood RNA tubes for storage can significantly improve the quality of RNA isolated from blood samples, but PAXgene whole blood RNA tubes are too expensive and the cost of sample collection is too high.

[0007] If samples cannot be extracted immediately after collection, they can be stored in RNA preservation solution. The main component of RNA preservation solution is a protein denaturant, which inhibits RNase activity to prevent RNA degradation. However, the effectiveness of existing preservation solutions is unstable. When samples need to be transported, repeatedly frozen and thawed, or stored for a long time, temperature fluctuations may affect the detection of RNA in the sample.

[0008] Summary of the Invention

[0009] In order to overcome the above-mentioned drawbacks, a low-cost RNA preservation solution is provided that can keep RNA in the sample stable under different storage time and temperature changes. Specifically,

[0010] In a first aspect, the present invention provides an RNA preservation solution, which includes a protein denaturant, a chelating agent, a reducing agent and a pH buffer. The pH of the preservation solution is 3.5-5.0. The protein denaturant is used to inhibit RNase activity, and the protein denaturant includes guanidine isothiocyanate and lithium chloride (LiCl).

[0011] Preferably, the pH is 3.8-5.0, and can be any value in the above range, such as 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, or 3.8.

[0012] Preferably, the molar mass ratio of guanidine isothiocyanate to LiCl is 1:(1-2), and the specific ratio may be 1:1.5.

[0013] More preferably, the protein denaturant further comprises a vanadyl ribonucleoside complex.

[0014] Preferably, the final concentration of the protein denaturant is 1-10 M. It can be any value within the above range, or any range, for example, 1.0, 1.5, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 4.2, 4.5, 4.8, 5.0, 5.2, 5.5, 5.8, 6.0, 6.3, 6.6, 6.8, 7.0, 7.2, 7.5, 7.8, 8.0, 8.5, 9.0, 9.5, 10.0 M.

[0015] More preferably, the protein denaturant comprises 1.5-3.0 M guanidine isothiocyanate and 1.5-6.0 M LiCl. Further preferably, the protein denaturant comprises 1.5-3.0 M guanidine isothiocyanate, 1.5-6.0 M LiCl and 5-20 mM vanadyl ribonucleoside complex.

[0016] In a specific embodiment, the protein denaturant comprises 2M guanidine thiocyanate and 3M LiCl.

[0017] In a specific embodiment, the protein denaturant comprises 2 M guanidine isothiocyanate, 3 M LiCl and 10 mM vanadyl ribonucleoside complex.

[0018] Preferably, the chelating agent comprises EDTA, dipotassium ethylenediaminetetraacetate and / or tripotassium ethylenediaminetetraacetate. More preferably, the chelating agent comprises EDTA.

[0019] Preferably, the final concentration of the chelating agent is 0.3-2.0 mM, which can be any value in the above range, or any range, such as 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 mM.

[0020] More preferably, the chelating agent comprises EDTA at a final concentration of 0.7-1.5 mM, and further preferably, the chelating agent comprises EDTA at a final concentration of 1.0 mM.

[0021] Preferably, the reducing agent includes one or more of tris(2-carboxyethyl)phosphine, β-mercaptoethanol and dithiothreitol.

[0022] More preferably, the reducing agent comprises β-mercaptoethanol.

[0023] Preferably, the final concentration of the reducing agent is 0.5%-3% by mass, more preferably, it can be any value in the above range, or any range, such as 0.5, 0.8, 1.0, 1.2, 1.5, 1.8, 2.0, 2.5, 3.0%.

[0024] More preferably, the reducing agent comprises 0.5%-2% of β-mercaptoethanol, preferably 0.8%-1.5%.

[0025] In a specific embodiment, the reducing agent comprises 1% β-mercaptoethanol.

[0026] Preferably, the RNA preservation solution further comprises a surfactant.

[0027] More preferably, the surfactant includes one or more of anionic surfactants, nonionic surfactants, cationic surfactants or amphoteric surfactants, such as Tween 20, SDS, Triton X-100, lithium dodecyl sulfate and the like.

[0028] Preferably, the final concentration of the surfactant is 0.2%-5.0% by mass, more preferably, it can be any value in the above range, or any range, for example, 0.2, 0.3, 0.4, 0.5, 0.8, 1.0, 1.2, 1.5, 1.8, 2.0, 2.5, 3.0, 3.5, 3.8, 4.0, 4.2, 4.5, 4.8, 5.0%.

[0029] Preferably, the RNA preservation solution further comprises salt ions, such as ammonium sulfate, sodium pyrophosphate, and the like.

[0030] More preferably, the final concentration of the salt ion is 5-30 mM, for example, 2.5-15 mM ammonium sulfate and 2.5-15 mM sodium pyrophosphate. It can be any value within the above range, or any range, for example 2.5, 3.0, 3.5, 4.0, 4.2, 4.5, 4.8, 5.0, 5.2, 5.5, 5.8, 6.0, 6.3, 6.6, 6.8, 7.0, 7.2, 7.5, 7.8, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0 mM.

[0031] Preferably, the RNA preservation solution further comprises glycine, and the final concentration of glycine is 30-70 mM, which can be any value in the above range, or any range, such as 30, 35, 40, 45, 50, 55, 60, 65, 70 mM.

[0032] Preferably, the pH buffer of the RNA preservation solution includes concentrated hydrochloric acid, Tris buffer, citrate buffer, sodium acetate buffer, phosphate buffer and HEPES, etc., to maintain the pH of the preservation solution at 3.5-5.0.

[0033] In a specific embodiment, the RNA preservation solution includes 2M guanidine thiocyanate, 3M LiCl, 1mM EDTA, 10mM ammonium sulfate, 1% Tween20, 10mM sodium pyrophosphate, 1% β-mercaptoethanol, 50mM glycine, the buffer is concentrated hydrochloric acid, the balance is H2O, and the pH is 4.8. Alternatively,

[0034] The RNA preservation solution includes 2M guanidine isothiocyanate, 3M LiCl, 1mM EDTA, 10mM vanadyl ribonucleoside complex, 10mM ammonium sulfate, 1% Tween20, 10mM sodium pyrophosphate, 1% β-mercaptoethanol, 50mM glycine, the buffer is concentrated hydrochloric acid, the balance is H2O, and the pH is 4.8.

[0035] The above percentage concentrations are percentages by mass.

[0036] In a second aspect, the present invention provides a method for preparing the above-mentioned RNA preservation solution, which comprises mixing various components of the RNA preservation solution, adjusting the pH with a pH buffer, and filtering to obtain the RNA preservation solution.

[0037] The various components of the RNA preservation solution are as defined above.

[0038] In a third aspect, the present invention provides a method for preserving a sample containing RNA, the method comprising preserving the sample using any of the above-mentioned RNA preservative solutions.

[0039] Preferably, the sample is derived from any sample, including but not limited to tissue, cells, body fluid, air, soil and / or surface of an object, etc. More preferably, the body fluid is derived from saliva, blood, serum, tissue fluid, urine, semen, amniotic fluid, cerebrospinal fluid, conjunctival fluid, vaginal fluid, feces or sweat, etc.

[0040] Further preferably, the sample is collected from a throat swab, sputum sample, alveolar lavage fluid sample, peripheral blood sample, plasma sample, serum sample, lymph node sample, gastrointestinal tissue sample, stool sample or urine sample, etc.

[0041] Preferably, the preservation method does not belong to a method for diagnosing and / or treating a disease.

[0042] Beneficial effects of the present invention:

[0043] 1. The RNA preservation solution of the present invention can be applied to different storage temperatures, such as 4°C, room temperature, and 37°C, which can avoid RNA damage caused by repeated freezing and thawing, and is conducive to storage under changing temperature conditions and long-distance transportation.

[0044] 2. The RNA preservation solution of the present invention can stably maintain the total amount of RNA in the sample and maintain the sample for a long period of time, up to at least 7 days. Even after 7 days of storage at 37°C, the amplification Ct value remains the same as the Ct value detected immediately at 4°C. This preservation solution has excellent stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 shows the concentration gradient amplification curve of PBGD using whole blood RNA.

[0046] Figure 2 shows the amplification curve of RT-qPCR after purification using the Qiagen virus kit.

[0047] FIG3 shows the RT-qPCR amplification curves of solution A and solution B after storage at three temperatures (1 is 4° C., 2 is room temperature, and 3 is 37° C.) for 24 h.

[0048] FIG4 shows the RT-qPCR amplification curves of solution A and solution B after storage at three temperatures (1 is 4° C., 2 is room temperature, and 3 is 37° C.) for 3 days.

[0049] FIG5 shows the RT-qPCR amplification curves of solution A and solution B after storage at three temperatures (1 is 4° C., 2 is room temperature, and 3 is 37° C.) for 7 days. DETAILED DESCRIPTION

[0050] The technical solutions of the present invention are further described below with reference to specific examples. It will be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The main features of the present invention can be applied to various embodiments without departing from the scope of the present invention. Those skilled in the art will recognize or be able to confirm that, using only routine experimentation, many equivalents can be applied to the specific steps described herein. These equivalents are considered to be within the scope of the present invention.

[0051] The instruments, reagents and consumables involved in the examples are as follows:

[0052] 1. Experimental instrument: ABI 7500 fluorescence quantitative PCR instrument

[0053] 2. Experimental reagents and consumables:

[0054] Table 1: Experimental reagents and consumables

[0055] Table 2: RNA Preservation Solution B

[0056] After preparation, the pH value was adjusted to 4.8 with concentrated hydrochloric acid and then filtered through a 0.22 μm filter membrane.

[0057] Example 1 Preservation effect of RNA preservation solution at different temperatures

[0058] Whole blood RNA was extracted using Takara RNAiso Blood and the following experiments were performed:

[0059] Experiment 1: RNA concentration gradient determination

[0060] 1-1: Detection steps

[0061] Whole blood RNA was divided into three concentration gradients of 10 ng, 1 ng, and 0.1 ng for RT-qPCR. Human internal reference genes were selected, and the reaction amplification system was prepared according to Table 3. The primer sequences were designed and synthesized. The primers are shown in Table 4, and the reaction conditions are shown in Table 5.

[0062] Table 3: Amplification system

[0063] Table 4: Primer probe sequences

[0064] Table 5: RT-qPCR reaction conditions

[0065] 1-2 Test results

[0066] Since a Ct value less than 35 is considered positive, we observed which concentration had a Ct value closer to 30. The results showed that when the total amount was 10 ng, the Ct value was around 28, the Ct value of 1 ng of whole blood RNA was around 30, and the Ct value of 0.1 ng of whole blood RNA was around 32. It can be seen that the Ct value between 0.1 ng and 10 ng was between 28 and 32, and the above final concentration range detection was feasible (see Figure 1, where the curves from left to right are 10 ng, 1 ng, and 0.1 ng (of which 0.1 ng was repeated in two batches), and the NoRT-10 ng group was in a horizontal state).

[0067] Experiment 2: RNA preservation solution storage and immediate detection

[0068] 2-1 Detection steps:

[0069] 60 ng of whole blood RNA was mixed into 140 μL of Solution A (Group A), Solution B (Group B), and 10 mM Tris (pH 8.0) (Group C). POS-CTR was used as a positive control (POS-CTR). RNA was extracted using the Qiagen Viral RNA Mini Kit according to the manufacturer's instructions and dissolved in 50 μL of DEPC water. 5 μL (approximately 1 ng of RNA) was used for RT-qPCR, following the same procedures as in Experiment 1.

[0070] 2-2 Test results:

[0071] The results are shown in Figure 2. The results show that the Ct values ​​of the three preservation solutions (A, B, and C) were around 30 when tested immediately after treatment. The three preservation solutions had the same effect on the RNA extraction efficiency. The Ct values ​​were slightly delayed compared to the positive control (POS-CTR), but were generally within an acceptable range (see Figure 2, where the curves from left to right are the preservation solution POS-CTR, A, B, and C treatment groups, repeated for two batches).

[0072] Experiment 3: RNA storage solution after storage at different temperatures and durations

[0073] 3-1 Detection steps:

[0074] Solution A (Group A) and Solution B (Group B) were each divided into three aliquots, each with a volume of approximately 640 μL. 320 ng of whole blood RNA (RNA concentration approximately 0.5 ng / μL) was added and stored at 4°C, room temperature, and 37°C, respectively. A positive control was also prepared in 10 mM TrisHCl (pH 8.0) (Group P) and frozen at -80°C.

[0075] At 1 day, 3 days, 7 days, and one month, 140 μL of the liquid from each tube was taken out to extract RNA using the Qiagen Viral RNA Mini Kit according to the instructions, and finally each tube was dissolved in 50 μL of DEPC water;

[0076] For RT-qPCR, 5 μL was used as a template and repeated twice; PBGD was used as the target and the probe was labeled with FAM. The operating conditions were the same as those in Experiment 1.

[0077] 3-2 Test results:

[0078] The RT-qPCR results after 24 hours of storage under three temperature conditions (1 is 4°C, 2 is room temperature, and 3 is 37°C, stored with liquid A and liquid B respectively) showed that the Ct value of liquid A (A1) stored at 4°C was delayed and was around 34, while the Ct values ​​of liquid B (B1) and the positive control (P) were basically the same, all around 30 (see the upper left figure of Figure 3, the three groups of curves from left to right (two for a group) are B1, P and A1); under room temperature and 37°C conditions, after 24 hours, liquid A (room temperature and 37°C different temperatures are respectively The amplification curves of solution B (labeled as A2 and A3 at different temperatures) no longer have peaks, while the Ct values ​​of solution B (labeled as B2 and B3 at different temperatures) and the positive control are basically the same, around 30 (see the upper right figure of Figure 3, the two groups of curves from left to right (two as a group) are B2 and P respectively; the lower left figure of Figure 3, the two groups of curves from left to right (two as a group) are B3 and P respectively); the negative control (NTC) has no peak (see the lower right figure of Figure 3); the above results show that the preservation solution of the present invention can stably preserve RNA at different temperatures.

[0079] Three days later, Solution A stored at 4°C still showed a slight peak, but the Ct value was relatively low (around 38). There was no amplification at room temperature and 37°C (horizontal lines in the upper right and lower left corners of Figure 4). However, the Ct values ​​of Solution B and the positive control remained the same at the three temperatures, around 30 (see Figure 4, with the same symbols as in Figure 3).

[0080] Seven days later, no amplification was observed after extraction from the RNA extracted from Solution A stored at the three temperature conditions (horizontal lines in the upper left, upper right, and lower left panels of Figure 5 ). The amplification Ct values ​​of Solution B and the positive control at the three temperature conditions remained the same, around 30 (see Figure 5 , with the same symbols as in Figure 3 ).

[0081] The above results show that even after storage at room temperature and 37°C for one week, the RNA preservation effect of the preservation solution of the present invention (solution B) is significantly better than that of solution A, can prevent RNA degradation, stably amplify RNA, and maintain a high Ct value, which is the same as that of the positive control.

[0082] The preservation solution obtained by removing the vanadyl ribonucleoside complex from solution B can also obtain results basically the same as those of solution B in Experiment 3.

[0083] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0084] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0085] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. An RNA preservation solution, characterized in that: The RNA preservation solution comprises a protein denaturant, a chelating agent, a reducing agent and a pH buffer. The pH of the preservation solution is 3.5-5.

0. The protein denaturant is used to inhibit the activity of RNase. The protein denaturant comprises guanidine isothiocyanate and lithium chloride (LiCl).

2. The RNA preservation solution according to claim 1, characterized in that The molar mass ratio of guanidine isothiocyanate to LiCl is 1:(1-2), preferably, the molar mass ratio of guanidine isothiocyanate to LiCl is 1:1.

5.

3. The RNA preservation solution according to any one of claims 1-2, characterized in that The final concentration of the protein denaturant is 1-10 M. Preferably, the protein denaturant includes 1.5-3.0 M guanidine thiocyanate and 1.5-6.0 M LiC.

4. The RNA preservation solution according to any one of claims 1 to 3, characterized in that The chelating agent includes EDTA, dipotassium ethylenediaminetetraacetate and / or tripotassium ethylenediaminetetraacetate, and the final concentration of the chelating agent is 0.3-2.0 mM.

5. The RNA preservation solution according to any one of claims 1 to 4, characterized in that The reducing agent comprises one or more of tris(2-carboxyethyl)phosphine, beta-mercaptoethanol and dithiothreitol, and the final concentration of the reducing agent is 0.5%-3% by mass.

6. The RNA preservation solution according to any one of claims 1 to 5, characterized in that The RNA preservation solution also includes a surfactant, and the final concentration of the surfactant is 0.2%-5.0% by mass.

7. The RNA preservation solution according to any one of claims 1 to 6, characterized in that The RNA preservation solution also includes salt ions, and the final concentration of the salt ions is 5-30 mM.

8. The RNA preservation solution according to any one of claims 1 to 7, characterized in that The protein denaturant further comprises a vanadyl ribonucleoside complex. Preferably, the protein denaturant comprises 1.5-3.0 M guanidine isothiocyanate, 1.5-6.0 M LiCl and 5-20 mM vanadyl ribonucleoside complex.

9. The method for preparing the RNA preservation solution according to any one of claims 1 to 8, characterized in that: The preparation method comprises mixing various components of the RNA preservation solution, adjusting the pH with a pH buffer, and filtering to obtain the RNA preservation solution.

10. A method for preserving a sample containing RNA, characterized in that: The storage method comprises storing the sample using the RNA storage solution described in any one of claims 1-8.

11. The storage method according to claim 10, characterized in that: The sample may be derived from tissue, cells, body fluid, air, soil and / or the surface of an object.

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