Novel liquid for preserving RNA (Ribonucleic Acid)

Abstract

The invention discloses a non-toxic RNase inhibition reagent for RNA preservation, the core of the non-toxic RNase inhibition reagent comprises an RNase inhibition component based on an ionic liquid and a natural choline cationic material, the concentration of the component is set to be 3%, exogenous and endogenous RNase with the concentration larger than or equal to 98.7% can be rapidly and efficiently inactivated, and the degradation effect of RNase on RNA is blocked from the source; meanwhile, the reagent has the remarkable advantages of no toxicity and no irritation, completely meets the application standard of a green laboratory, and has good compatibility with mainstream RNA extraction processes of a Trizol method, a column method, a paramagnetic particle method and the like; the reagent is preloaded at a sample collection starting point and carries out immediate protection, so that an RNA sample can be effectively preserved at 4 DEG C or 25 DEG C, and the success rate of RNA extraction is increased by more than or equal to 40%.

Description

Technical Field

[0001] This invention belongs to the field of biological RNA sample preservation technology, and particularly relates to a compositional protectant for preserving RNA in liquid samples at room temperature and its application method. Background Technology

[0002] Prostate cancer is one of the most common cancers in men. Whether a patient has prostate cancer is primarily determined by PSA, a cancer factor in the blood. However, due to a diagnostic accuracy rate as low as 30%, a significant number of patients require additional invasive biopsies, resulting in side effects such as bleeding and pain.

[0003] As a non-invasive method, urine-based diagnostic testing is convenient for patients, eliminating the need for invasive biopsies and thus diagnosing cancer without side effects. However, urine has a low concentration of nucleic acids, with even less of the prostate-derived components, particularly prostate-specific RNA. In most cases, due to various reasons, RNA cannot be immediately detected after extraction from biological samples. Once isolated from the body, RNA-containing biological samples become highly unstable and easily degraded. Preservation in liquid nitrogen or at -80°C typically limits the application of urine RNA detection. Therefore, developing a urine RNA preservation solution that effectively prevents RNA degradation and does not affect subsequent test results is crucial for the molecular biological detection of urine RNA and has significant implications for expanding the applications of urine sample testing.

[0004] Current RNA preservation products have the following shortcomings: 1. Most patents target samples with high RNA content, such as tissues, blood, and swabs, while preservation solutions for body fluids with low RNA content, such as urine, are scarce. 2. Existing urine RNA preservation agents, such as Norgen's urine preservation tubes, require RNA extraction from the mixture after urine preservation. Conventional extraction kits extract samples in volumes of only 2-5 ml per batch, and the extraction cost is high. There is an urgent need to develop a urine RNA preservation agent that allows for the enrichment of the precipitate through low-speed centrifugation, followed by RNA extraction from the precipitate using the conventional Trizol method. This would easily achieve single-batch extraction of tens of milliliters of urine sample, significantly reducing extraction costs. 3. Existing urine RNA preservation agents primarily use β-mercaptoethanol and dithiothreitol (DTT) as reducing agents. These reducing agents are used to reduce disulfide bonds in proteins, preventing intramolecular or intermolecular disulfide bonds formed between cysteine ​​residues. However, β-mercaptoethanol itself only has one sulfhydryl group, and its effect generally only lasts for 2-3 days. Therefore, β-mercaptoethanol needs to be added every 2-3 days in experiments to maintain its effect. However, β-mercaptoethanol is volatile, highly irritating, and toxic. Dithiothreitol (DTT) has two thiol groups and its reducing power is slightly stronger than β-mercaptoethanol. Its effect can last for 3-7 days, but it often cannot reduce disulfide bonds embedded within the protein structure (inaccessible to solvents). Reduction of these disulfide bonds often requires prior protein denaturation (high-temperature heating or the addition of denaturing agents such as 6M guanidine hydrochloride, 8M urea, or 1% SDS). Furthermore, DTT is easily oxidized by air, and its stability is poor, requiring cryopreservation or treatment in an inert gas atmosphere to extend its shelf life. Therefore, a more stable and effective reducing agent needs to be found. 4. Existing research on preservatives for urinary RNA mainly verifies changes in total RNA content and differences in mRNA amplification, lacking data on the size distribution of urinary RNA fragments and RNA-Seq validation data. Clearly defining the size range of RNA fragments and obtaining RNA-Seq validation data are crucial for RNA sequencing applications.

[0005] Furthermore, clinical testing encounters various types of urine samples, ranging from exceptionally clear and transparent samples with few shed cells to samples showing hematuria. This complexity increases the difficulty of preserving urine RNA, a fact reflected in the actual exploration process of this invention. The optimal dosages of dissociation agents, sedimentation aids, and reducing agents vary depending on the sample condition. Extensive testing on numerous samples is necessary to obtain a protective agent applicable to various types of urine samples, comprehensively balancing their protective effects on different samples to meet the needs of practical clinical applications. Summary of the Invention

[0006] The primary objective of this invention is to provide an RNA preservative that effectively maintains the stability of RNA in transported and preserved liquid samples, especially urine samples. More importantly, it is applicable to the preservation of various types of urine samples. Urine samples preserved with the preservative solution of this invention can be enriched by low-speed centrifugation, and then RNA can be extracted from the precipitate using the conventional Trizol method. This provides a convenient method for downstream extraction, increases the extraction volume of the original urine sample, solves the problem of difficult large-volume urine sample extraction, and greatly reduces the extraction difficulty and cost. This invention achieves the problem of room temperature preservation and transportation of large-volume urine samples, and the extracted RNA is well-suited for subsequent RT-PCR and RNA-seq applications. Furthermore, various methods have verified that the RNA preserved with this preservative solution exhibits excellent properties such as high concentration, high integrity, and high stability after long-term storage.

[0007] This invention is achieved using the following technical solution:

[0008] An RNA protectant comprising the following components: a dissociation agent, a settling agent, a reducing agent, and a nuclease inhibitor; wherein:

[0009] The liquid release agent includes at least one of guanidine isothiocyanate and guanidine hydrochloride;

[0010] The reducing agents include: tris(2-carboxyethyl)phosphonic acid hydrochloride;

[0011] The flocculants include at least one of PEG and sulfosalicylic acid.

[0012] The concentration of the RNA protectant in the protectant is 3-5M, preferably 3.5-4.5M, more preferably 3.8-4.2M, and most preferably 4M.

[0013] The RNA protectant, tris(2-carboxyethyl)phosphonic acid hydrochloride, is present in a concentration of 50-300 mM, preferably 150-250 mM, more preferably 180-220 mM, and most preferably 200 mM.

[0014] The RNA protectant has the following concentrations: PEG in the protectant is 5-30%, preferably 15-25%, more preferably 18-22%, and most preferably 20%; sulfosalicylic acid in the protectant is 250mM-1M, preferably 300-800mM, more preferably 400-600mM, and most preferably 500mM.

[0015] The RNA protectant, a nuclease inhibitor, comprises one or more of the following components: ethylenediaminetetraacetic acid (EDTA) and its salts, ethylene glycol tetraacetic acid (EGTA) and its salts, aurantium tricarboxylic acid (ATA), glyceraldehyde, NaF, formamide, vanadium ribonucleoside complex, 8-hydroxyquinoline, bentonite, sodium dodecyl sulfate (SDS), and cysteine. The concentration of EDTA in the protectant is 5-100 mM, preferably 30-60 mM, more preferably 45-50 mM, and most preferably 50 mM.

[0016] The concentration of EGTA in the protective agent is 5mM-100mM, preferably 30-60mM, further preferably 45-50mM, and most preferably 50mM;

[0017] The concentration of ATA in the protective agent is 0.5-10 mM, preferably 2-8 mM, more preferably 4-6 mM, and most preferably 5 mM;

[0018] The concentration of glyceraldehyde in the protective agent is 50 mM-200 mM, preferably 60-150 mM, further preferably 80-120 mM, and most preferably 100 mM;

[0019] The concentration of NaF in the protective agent is 1 mg / ml-10 mg / ml, preferably 3-6 mg / ml, more preferably 4-5 mg / ml, and most preferably 5 mg / ml;

[0020] The concentration of formamide in the protective agent is 10 mg / ml-100 mg / ml, preferably 30-60 mg / ml, more preferably 40-50 mg / ml, and most preferably 50 mg / ml;

[0021] The concentration of the vanadium-based ribonucleoside complex in the protective agent is 10 mg / ml-100 mg / ml, preferably 30-60 mg / ml, more preferably 40-50 mg / ml, and most preferably 50 mg / ml;

[0022] The concentration of 9-hydroxyquinoline in the protective agent is 0.5%-5%, preferably 1%-5%, more preferably 1%-2%, and most preferably 2%.

[0023] The concentration of bentonite in the protective agent is 10 mg / ml-100 mg / ml, preferably 30-60 mg / ml, more preferably 40-50 mg / ml, and most preferably 50 mg / ml;

[0024] Sodium dodecyl sulfate (SDS) is present in the protective agent at a concentration of 0.5%-5%, preferably 1%-3%, more preferably 1%-2%, and most preferably 1%.

[0025] The concentration of cysteine ​​in the protective agent is 5-25 mM, preferably 10-25 mg / ml, more preferably 15-20 mg / ml, and most preferably 20 mg / ml;

[0026] The RNA protectant also includes: buffer solution and solvent.

[0027] The aforementioned RNA protectant,

[0028] The buffer solution includes one or more of the following components: citrate-sodium citrate buffer, Tris-HCl buffer, and HEPES buffer.

[0029] The solvent is sterile purified water.

[0030] The RNA protectant comprises: citrate-sodium citrate buffer with a pH range of 4.0-6.5, preferably pH 4.5, and a concentration of 0.1M-0.2M, preferably 0.1M; Tris-HCl buffer with a pH range of 6.0-7.0, preferably pH 6.8, and a concentration of 0.1M-0.2M, preferably 0.1M; and HEPES buffer with a pH range of 6.5-8.0, preferably pH 7.0, and a concentration of 0.1-0.2M, preferably 0.1M.

[0031] The RNA protectant further includes: lower alcohols, preferably including one or more of the following components: methanol, ethanol, and isopropanol.

[0032] The RNA protectant contains 10-40% lower alcohol, preferably 10-30%, more preferably 15-25%, and most preferably 20%.

[0033] The RNA protectant further includes a surfactant, preferably comprising one or more of the following components: Tween 20, Triton X-100, Nonidet P40, and Brij35.

[0034] The RNA protectant contains a surfactant in a content of 5-20%, preferably 5-15%, more preferably 8-12%, and most preferably 10%.

[0035] A second object of the present invention is to provide the application of the aforementioned RNA protectant for preserving RNA in liquid samples, particularly RNA in urine samples.

[0036] Furthermore, the RNA protectant preserves urine at a temperature range of -20°C to 37°C, preferably 4°C to 37°C.

[0037] Furthermore, the collected urine was added to the RNA protectant.

[0038] Furthermore, the volume ratio of urine to RNA protectant is 3-5:1, preferably 4:1.

[0039] The beneficial effects of this invention.

[0040] Advantage 1: It can be used to test large batches of different types of clinical urine samples and can preserve RNA from various urine samples.

[0041] Advantage 2: The flocculant used in this invention enriches RNA by precipitating ribonucleoproteins. Previously reported flocculants for pure nucleic acids, such as glycogen and LiCl, have not been effective. This invention uniquely precipitates ribonucleoproteins by precipitating proteins, thereby precipitating RNA within the ribonucleoproteins. It has identified a flocculant and its optimal concentration ratio that is particularly suitable for enriching ribonucleoproteins in urine samples.

[0042] Advantage 3: The RNA preserved in the preservation solution of this invention can be enriched and precipitated to the bottom of the tube by centrifugation. After removing the supernatant, the RNA in the precipitate can be extracted. This solves the problem of large extraction volumes in urine samples with low RNA content, which limits the extraction kit and increases extraction costs. For example, this invention can use a 50ml centrifuge tube to enrich and precipitate 50ml of urine, and then easily extract high-concentration RNA using the conventional Trizol method. In contrast, other urine preservation solutions, such as Norgen's urine preservation tubes, can only extract 5ml or more of urine samples using extraction reagents specifically designed for extracting large volumes of urine RNA, and the amount of RNA that can be extracted is very limited.

[0043] Advantage 4: The reducing agent used is not dithiothreitol (DTT) or β-mercaptoethanol, but tris(2-carboxyethyl)phosphine hydrochloride (TCEP). TCEP has a stronger reducing power than both β-mercaptoethanol and DTT, and its effect can last for 2-3 weeks.

[0044] Advantage 5: The evaluation of RNA preservation effect of the preservation solution in this invention not only assesses RNA concentration and RT-PCR application effect, but also RNA integrity and RNA-Seq application effect, providing more comprehensive verification data and facilitating the application of downstream technologies.

[0045] Advantage 6: It can be stored at room temperature for up to 20 days with excellent stability; it solves the problem of sample preservation and transportation after clinical sampling. Attached Figure Description

[0046] Figure 1 The image shows the Qsep100 fragment analysis of RNA extracted from pure urine on day 0 in Example 9.

[0047] Figure 2The image shows the RNA Qsep100 fragment extracted from urine preserved in RNA preservation solution for 10 days in Example 9.

[0048] Figure 3 The image shows the RNA Qsep100 fragment extracted from urine after 20 days of preservation in RNA preservation solution in Example 9. Detailed Implementation

[0049] The following examples are intended to further illustrate the present invention, but not to limit it.

[0050] Example 1: Synergistic effect of separation agent and settling aid in this preservation solution

[0051] Collect morning urine samples from healthy individuals. After collection, add 16 ml of urine to a 50 ml centrifuge tube / purified water containing the preservation solution listed below. The volume ratio of purified water or preservation solution to urine is 1:4. Incubate at room temperature (23.6℃) using sterile purified water as the solvent.

[0052] Table 1

[0053] Group number Components 1 Purified water, 0-day enrichment and precipitation 2 Purified water, left at room temperature for 5 days 3 2M Guanidine Hydrochloride 4 4M Guanidine Hydrochloride 5 8M Guanidine Hydrochloride 6 2M Guanidine Isothiocyanate 7 4M Guanidine Isothiocyanate

[0054] 8 6M Guanidine Isothiocyanate 9 20% PEG 10 30% PEG 11 40% PEG 12 2M guanidine hydrochloride, 20% PEG 13 4M Guanidine Hydrochloride, 20% PEG 14 8M guanidine hydrochloride, 20% PEG 15 2M guanidine isothiocyanate, 20% PEG 16 4M Guanidine Isothiocyanate, 20% PEG 17 6M Guanidine Isothiocyanate, 20% PEG

[0055] In Group 1, urine samples collected with purified water were immediately enriched and precipitated. The precipitate was then mixed with RZ reagent from the Tiangen Total RNA Extraction Kit (DP419) and stored at -80°C for 5 days before total RNA extraction. In Group 2, urine samples collected with purified water were stored at room temperature for 5 days, then enriched and precipitated together with other preservation solutions before RNA extraction. For Groups 3-17, urine samples containing preservation solutions were stored at room temperature for 5 days, then centrifuged at 4000g for 30 min to enrich the precipitate and extract RNA. Total RNA was extracted using the Tiangen Total RNA Extraction Kit (DP419). Two-step RT-PCR was used to detect ACTB and PSA gene mRNA. The results are shown in Table 2.

[0056] Table 2

[0057]

[0058]

[0059] The results of this embodiment show that after 5 days of standing at room temperature, the urine in the purified water group showed bacterial growth and complete RNA degradation. The solutions in the groups with added guanidine hydrochloride and guanidine isothiocyanate became clear, indicating that guanidine salts inhibited bacterial growth. Groups 13 (4M guanidine hydrochloride, 20% PEG) and 16 (4M guanidine isothiocyanate, 20% PEG) showed the best results. Too high a guanidine salt concentration (8M guanidine hydrochloride or 6M guanidine isothiocyanate) resulted in no flocculent precipitate at the bottom of the tube, preventing RNA enrichment. Too low a concentration (2M) failed to inhibit RNase activity, leading to RNA degradation. By combining an appropriate concentration of guanidine salt with PEG, the guanidine salt inhibited RNase activity without denaturing ribonucleoproteins and releasing RNA, while PEG enhanced the precipitation effect, resulting in more RNA. This embodiment provides a new enrichment approach: enriching ribonucleoprotein complexes rather than simply nucleic acids, allowing for the acquisition of RNA-containing ribonucleoprotein complex precipitates under low-speed centrifugation at 4000g.

[0060] However, the best preservation solutions in this embodiment, Group 13 (4M guanidine hydrochloride, 20% PEG) and Group 16 (4M guanidine isothiocyanate, 20% PEG), still have a certain gap compared with the results of Group 1's urine on day 0 plus pure water. Their optimization and improvement will be reflected in subsequent embodiments.

[0061] Example 2: Nuclease Inhibitor Test

[0062] Collect morning urine samples from healthy individuals. After collection, add 16 ml of urine to a 50 ml centrifuge tube / purified water containing the preservation solution listed below. The volume ratio of purified water or preservation solution to urine is 1:4. Incubate at room temperature (23.4℃) using sterile purified water as the solvent.

[0063] Table 3

[0064]

[0065] In Group 1, urine samples collected with purified water were immediately enriched and precipitated. The precipitate was then treated with RZ reagent from the Tiangen Total RNA Extraction Kit (DP419) and stored at -80°C for 5 days before total RNA extraction. In Group 2, urine samples collected with purified water were stored at room temperature for 5 days, then enriched and precipitated together with other preservation solutions before RNA extraction. For Groups 3-17, urine samples containing preservation solutions were stored at room temperature for 5 days, then centrifuged at 4000g for 30 min to enrich the precipitate and extract RNA. Total RNA was extracted using the Tiangen Total RNA Extraction Kit (DP419). Two-step RT-PCR was used to detect ACTB and PSA gene mRNA. The results are shown in Table 4.

[0066] Table 4

[0067]

[0068]

[0069] The results of this embodiment show that simply adding nuclease inhibitors is ineffective. Adding nuclease inhibitors to the optimal preservation solution from Example 1 can appropriately improve RNA protection; however, there was no significant difference in the effects of several nuclease inhibitors.

[0070] Example 3: Reducing Agent Test

[0071] Based on Group 3 (4M guanidine hydrochloride, 20% PEG, 50mM EDTA) and Group 4 (4M guanidine isothiocyanate, 20% PEG, 50mM EDTA) in Example 2, it is expected that the RNA yield will be improved by adding a reducing agent.

[0072] Collect morning urine samples from healthy individuals. After collection, add 16 ml of urine to a 50 ml centrifuge tube / purified water containing the preservation solution listed below. The volume ratio of purified water or preservation solution to urine is 1:4. Incubate at room temperature (24.1℃) using sterile purified water as the solvent.

[0073] Table 5

[0074] Group number Components 1 Purified water, 0-day enrichment and precipitation 2 Purified water, left at room temperature for 5 days 3 4M guanidine hydrochloride, 20% PEG, 50mM EDTA 4 4M guanidine isothiocyanate, 20% PEG, 50mM EDTA 5 4M guanidine hydrochloride, 20% PEG, 50mM EDTA, 8% β-mercaptoethanol 6 4M guanidine isothiocyanate, 20% PEG, 50mM EDTA, 8% β-mercaptoethanol 7 4M guanidine hydrochloride, 20% PEG, 50mM EDTA, 200mM M DTT 8 4M guanidine isothiocyanate, 20% PEG, 50mM EDTA, 200mM M DTT 9 4M guanidine hydrochloride, 20% PEG, 50mM EDTA, 200mM MTCEP 10 4M guanidine isothiocyanate, 20% PEG, 50mM EDTA, 200mM MTCEP 11 8% β-mercaptoethanol 12 200mMDTT 13 200mM TCEP

[0075] In Group 1, urine samples collected with purified water were immediately enriched and precipitated. The precipitate was then treated with RZ reagent from the Tiangen Total RNA Extraction Kit (DP419) and stored at -80°C for 5 days before total RNA extraction. In Group 2, urine samples collected with purified water were stored at room temperature for 5 days, then enriched and precipitated together with other preservation solutions before RNA extraction. For Groups 3-13, urine samples containing preservation solutions were stored at room temperature for 5 days, then centrifuged at 4000g for 30 min to enrich the precipitate and extract RNA. Total RNA was extracted using the Tiangen Total RNA Extraction Kit (DP419). Two-step RT-PCR was used to detect ACTB and PSA gene mRNA. The results are shown in Table 6.

[0076] Table 6

[0077]

[0078]

[0079] The results of this embodiment show that simply adding reducing agents such as β-mercaptoethanol, DTT, and TCEP yields poor results. Adding reducing agents to the optimal preservation solution from Example 2 improves RNA protection, with DTT and TCEP showing better results after 5 days of storage at room temperature. TCEP exhibits good stability and solubility in aqueous solutions. It also shows good stability in acidic and alkaline solutions. Tests on the stability and duration of action of several reducing agents will be presented in subsequent embodiments.

[0080] Example 4: Stability and reaction time test of reducing agent

[0081] Collect morning urine samples from healthy individuals. After collection, add 16 ml of urine to 50 ml centrifuge tubes containing the following preservation solution / purified water. The volume ratio of purified water or preservation solution / purified water to urine is 1:4. Incubate at room temperature (23.8℃) using sterile purified water as the solvent.

[0082] For Group 1, urine samples collected with purified water were immediately enriched and precipitated. The precipitate was then treated with RZ reagent from the Tiangen Total RNA Extraction Kit (DP419) and stored at -80°C for 5 days before total RNA extraction. For Groups 2-21, samples treated with purified water or preservation solution were stored at room temperature for 3, 6, 10, 20, and 30 days, followed by centrifugation at 4000g for 30 min to enrich the precipitate and extract RNA. The Tiangen Total RNA Extraction Kit (DP419) was used to extract total RNA from urine. Two-step RT-PCR was used to detect ACTB and PSA gene mRNA. The results are shown in Table 7.

[0083] Table 7

[0084]

[0085]

[0086] Based on the results of this embodiment, the effect of β-mercaptoethanol can only be maintained for 3 days, and the effect decreases significantly after 6 days of storage; the effect of DTT can be maintained for 6 days, and the effect decreases significantly after 10 days of storage; the effect of TCEP is relatively stable up to 20 days, and the effect decreases slightly after 30 days. Therefore, TCEP is selected as the reducing agent in the preservation solution of this invention.

[0087] Example 5: Testing of different urine samples

[0088] Prepare the following components:

[0089] The solvent is sterile pure water.

[0090] Table 8

[0091]

[0092] Eight urine samples were collected from healthy individuals (NM group), eight urine samples from individuals with prostate cancer (PCA group), eight urine samples from individuals with bladder cancer (BC group), and eight urine samples from individuals with bladder cancer (BCB group). After collection, 16 ml of each sample was added to centrifuge tubes containing control reagents and preservation solution, respectively. The volume ratio of purified water / preservation solution to urine was 1:4. The room temperature was 23.8℃.

[0093] Urine samples added to the control reagent were enriched and precipitated immediately on day 0. The precipitate was then treated with RZ reagent from the Tiangen Total RNA Extraction Kit (DP419) and stored at -80°C for 5 days before total RNA extraction. Urine samples containing the preservation solution were stored at room temperature for 5 days before total RNA extraction using the Tiangen Total RNA Extraction Kit (DP419). PSA gene mRNA was detected using a two-step RT-PCR method. The results are shown in Table 9.

[0094] Table 9

[0095]

[0096]

[0097]

[0098] The results showed that urine samples with a high number of exfoliated cells exhibited significant precipitation enrichment after centrifugation, with little difference compared to the day 0 control group. However, urine samples with very few exfoliated cells showed almost no visible precipitation after centrifugation, resulting in a significant difference compared to the day 0 control group. This suggests that the preservation solution formulation needs to be optimized for different urine sample types to ensure that samples with few exfoliated cells can achieve results comparable to the control group, thus meeting the needs of various clinical sample types. This optimization will be demonstrated in subsequent examples.

[0099] Example 6: Screening Test of Settling Agents

[0100] Based on the results of Example 5, this example aims to improve the precipitation enrichment effect by screening various flocculants in order to meet the needs of samples with fewer detached cells.

[0101] Two morning urine samples (with a high number of exfoliated cells) from healthy individuals were collected, designated A and B; two random urine samples (with fewer exfoliated cells) from healthy individuals were collected, designated C and D. After collection, 16 ml of urine from each sample was added to a 50 ml centrifuge tube containing either purified water or preservation solution as specified below. The volume ratio of purified water or preservation solution to urine was 1:4. The temperature was maintained at 24.6℃.

[0102] Table 10

[0103]

[0104]

[0105] In Group 1, urine samples collected with purified water were immediately enriched and precipitated. The precipitate was then mixed with RZ reagent from the Tiangen Total RNA Extraction Kit (DP419) and stored at -80°C for 5 days before total RNA extraction. In Group 2, urine samples collected with purified water were stored at room temperature for 5 days, then enriched and precipitated together with other preservation solutions before RNA extraction. For Groups 3-15, urine samples containing preservation solutions were stored at room temperature for 5 days, then centrifuged at 4000g for 30 min to enrich the precipitate and extract RNA. Total RNA was extracted using the Tiangen Total RNA Extraction Kit (DP419). A two-step RT-PCR method was used to detect PSA gene mRNA. The results are shown in Table 11.

[0106] Table 11

[0107]

[0108]

[0109] The results showed that sulfosalicylic acid and PEG had better precipitation-aiding effects. For the two samples with fewer detached cells, group 13 (4M guanidine isothiocyanate, 50mM EDTA, 200mM TCEP, 20% PEG, 500mM sulfosalicylic acid, 20% isopropanol) showed the best effect. The precipitation-aiding effect of agents capable of precipitating proteins was better than that of agents capable only of precipitating nucleic acids. This demonstrates that the protective agent of this invention is most effective when precipitating ribonucleoproteins.

[0110] Example 7: Preservative Solution Optimization Test

[0111] Based on Example 6, Group 13 (4M guanidine isothiocyanate, 50mM EDTA, 200mM TCEP, 20% PEG, 500mM sulfosalicylic acid, 20% isopropanol), this example, for samples with fewer detached cells, adds the testing of surfactants and buffers, based on the fact that surfactants can reduce the adsorption of nucleic acid and protein molecules on the tube wall and buffers can maintain the stability of the solution.

[0112] Two morning urine samples (with a high number of exfoliated cells) from healthy individuals were collected, designated A and B; two random urine samples (with fewer exfoliated cells) from healthy individuals were collected, designated C and D. After collection, 16 ml of urine from each sample was added to a 50 ml centrifuge tube containing either purified water or preservation solution as specified below. The volume ratio of purified water or preservation solution to urine was 1:4. The temperature was maintained at 24.5℃.

[0113] Table 12

[0114]

[0115]

[0116] In Group 1, urine samples collected with purified water were immediately enriched and precipitated. The precipitate was then mixed with RZ reagent from the Tiangen Total RNA Extraction Kit (DP419) and stored at -80°C for 5 days before total RNA extraction. In Group 2, urine samples collected with purified water were stored at room temperature for 5 days, then enriched and precipitated together with other preservation solutions before RNA extraction. For Groups 3-10, urine samples containing preservation solutions were stored at room temperature for 5 days, then centrifuged at 4000g for 30 min to enrich the precipitate and extract RNA. The extraction reagent was the Tiangen Total RNA Extraction Kit (DP419) for total RNA extraction. A two-step RT-PCR method was used to detect PSA gene mRNA. The results are shown in Table 13.

[0117] Table 13

[0118]

[0119] The results showed that the results of various surfactants were slightly better than those of the group without surfactants, and the buffering environment was more effective under acidic and neutral conditions.

[0120] Example 8: Temperature range test of preservation solution for urine preservation

[0121] Prepare a urine sample RNA preservation solution, controlling the content of each component as follows: 4M guanidine isothiocyanate, 50mM EDTA, 200mM TCEP, 20% PEG, 500mM sulfosalicylic acid, 20% isopropanol, 10% Tween 20, and 0.1M citrate-sodium citrate buffer (pH 4.5). Inject 2 mL of the prepared preservation solution into vacuum urine collection tubes containing 8 mL of urine, inverting thoroughly 8-10 times to prepare 12 tubes. Store at -20℃, 4℃, room temperature, and 37℃ for 20 days, with 3 replicates per group. Separately, prepare two tubes of 8 mL pure urine with 2 mL of sterile purified water. Collect the precipitate on day 0 and extract total RNA from the urine using the Trizol method, storing at -80℃. Samples from groups treated with RNA preservation solution were collected at 10 and 20 days, and total urinary RNA was extracted using the Trizol method and stored at -80℃. The extraction reagent was the Tiangen Total RNA Extraction Kit (DP419). PSA gene mRNA was detected using a two-step RT-PCR method. The detection results (ct values) are shown in the table below.

[0122] Table 14

[0123] Serial Number Day 0 control group -20℃ 4℃ room temperature 37℃ 1 25.84 25.90 25.94 26.13 26.11 2 26.21 25.94 26.02 25.91 25.89 3 25.97 25.88 25.79 25.87 26.23

[0124] The results showed that the preservation solution was used for urine preservation at temperatures ranging from -20℃ to 37℃, with no significant difference in effectiveness compared to the 0-day control group.

[0125] Example 9: RNA integrity test and RNA-seq result evaluation

[0126] Prepare a urine sample RNA preservation solution, controlling the content of each component as follows: 4M guanidine isothiocyanate, 50mM EDTA, 200mM TCEP, 20% PEG, 500mM sulfosalicylic acid, 20% isopropanol, 10% Tween 20, and 0.1M citrate-sodium citrate buffer (pH 4.5). Inject 2mL of the prepared preservation solution into vacuum urine collection tubes containing 8mL of urine, inverting thoroughly 8-10 times to prepare two tubes. Store at room temperature (25℃±5℃) for 10 and 20 days, respectively. Separately, prepare one tube of 8mL pure urine with 2mL sterile purified water. Collect the precipitate on day 0 and extract total RNA using the Trizol method, storing at -80℃. Collect the precipitate from each group of samples with the added RNA preservation solution on days 10 and 20, extract total RNA using the Trizol method, and store at -80℃. All RNA fragments were analyzed using a Qsep100 fragment analyzer, and the results are as follows: Figure 1 As shown in Figures 2 and 3; the extracted RNA was used for library construction and sequencing, and the results are shown in Table 15:

[0127] Table 15

[0128]

[0129]

[0130] Example 9 tested the fragment size of extracted RNA. The results showed that the RNA fragment distribution after 10 and 20 days of preservation was almost identical to that of the pure urine sample from day 0. The RNA-Seq results showed no significant difference from the control group containing pure urine from day 0. This indicates that the preservation solution can ensure the integrity of RNA and provides reliable RNA sequencing results.

[0131] Example 10: Comparative Test with Norgen Urine Preservation Tubes

[0132] A urine sample RNA preservation solution was prepared as the preservation solution test group (DDTN group) of this invention, with the following component contents controlled: 4M guanidine isothiocyanate, 50mM EDTA, 200mM TCEP, 20% PEG, 500mM sulfosalicylic acid, 20% isopropanol, 10% Tween 20, and 0.1M citrate-sodium citrate buffer (pH 4.5). Norgen urine preservation tubes were purchased from Norgen Biotek Corp.'s Urine Collection and Preservation Tube (Cat. 18120). Three groups were set up: the DDTN group, the Norgen precipitate group, and the Norgen mixture group. After adding 8 ml of urine to each group and storing for 20 days, the DDTN group was centrifuged at 6000g for 20 min according to the sample pretreatment method of this invention, and the precipitate was retained. Total RNA was extracted from the urine using the Tiangen Total RNA Extraction Kit (DP419). The Norgen precipitate group was also centrifuged at 6000g for 20 min according to the sample pretreatment method, and the precipitate was retained. Total RNA was extracted from the urine using the Tiangen Total RNA Extraction Kit (DP419). The Norgen mixture group was directly extracted using the recommended method, with the entire 8 ml mixture in the storage tube extracted directly (using the Zymo Quick-cfRNA extraction kit). TM The RNA extracted using the Serum & Plasma Kit (R1059) was subjected to RT-PCR with triple replicates. The results are as follows:

[0133] Table 16

[0134]

[0135] Based on the test results, the DDTN precipitation group showed the best extraction effect. The extraction method using the Norgen urine preservation tube after enrichment precipitation was less effective. RNA could only be obtained by extracting a large volume of the mixture using the method recommended by the preservation solution itself. The extraction kit used in this method costs more than 10 times that of the total RNA extraction kit.

[0136] After removing rRNA from the RNA extracted from the above three groups, library construction and sequencing were performed. The rRNA removal reagent used was Hieff. The results of the One-Step rRNA Removal Kit (Cat No. 12258) are shown in the table below:

[0137] Table 17

[0138]

[0139]

[0140] RNA-Seq results showed that the mapping rate of the Norgen precipitate group and the Norgen mixture group was significantly lower than that of the DDTN group, indicating that the DDTN preservation solution was more effective than the Norgen preservation tube in preserving human RNA in urine. The unmapped reads (data not aligned to the human transcriptome) were mostly aligned to the microbial genome by bioinformatics analysis, indicating that there was a large amount of microbial contamination in the Norgen precipitate group and the Norgen mixture group, and their antibacterial effect on urine samples was not as good as that of DDT.

Claims

1. A non-toxic RNase inhibitor for RNA preservation, characterized in that, It contains an RNase inhibitory component based on ionic liquids and natural choline-based cationic materials, wherein the concentration of the inhibitory component is 3%, and it can rapidly inactivate ≥98.7% of exogenous and endogenous RNases.

2. The non-toxic RNase inhibitor for RNA preservation according to claim 1, characterized in that, The reagents are non-toxic and non-irritating, and meet the standards for green laboratories.

3. The non-toxic RNase inhibitor for RNA preservation according to claim 1, characterized in that, The reagent is compatible with at least one of the following RNA extraction methods: Trizol, column method, and magnetic bead method.

4. A method for preserving RNA samples, characterized in that, Using the non-toxic RNase inhibitor described in any one of claims 1-3, the RNA sample is pre-loaded at the start of sample collection and immediately protected.

5. The preservation method according to claim 4, characterized in that, Storing RNA samples at 4℃ or 25℃ increases the RNA extraction success rate by ≥40%.

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