CN114410739A

CN114410739A - RNA protective agent and application thereof

Info

Publication number: CN114410739A
Application number: CN202111422580.XA
Authority: CN
Inventors: 徐根明; 汤链; 王永利; 鞠巍; 冯金儒; 贺田芬; 刘雅静
Original assignee: Hunan Yearth Biotechnological Co ltd
Current assignee: Hunan Yearth Biotechnological Co ltd
Priority date: 2021-11-26
Filing date: 2021-11-26
Publication date: 2022-04-29

Abstract

The invention discloses an RNA protective agent and application thereof. The protective agent comprises the following components: chaotropic agents, precipitating agents, reducing agents, nuclease inhibitors, surfactants, lower alcohols, buffers and solvents. Wherein: the chaotropic agent is guanidine isothiocyanate or guanidine hydrochloride; the reducing agent is tris (2-carboxyethyl) phosphine hydrochloride; the settling agent is at least one of PEG and sulfosalicylic acid. The protective agent can effectively keep the stability of RNA in the transported and stored urine samples, more importantly, can ensure the stability of RNA in the normal-temperature transportation process, and is suitable for storing various clinical complicated urine samples. The urine sample preserved by the preserving fluid can be enriched and precipitated in a low-speed centrifugation mode, and then RNA in the precipitate is extracted by a conventional Trizol method, so that a convenient and fast mode is provided for downstream extraction, the extraction volume of the original urine sample is increased, the problem of difficulty in large-volume extraction of the urine sample is solved, the extraction difficulty and the extraction cost are greatly reduced, and the preserving fluid has a good application prospect.

Description

RNA protective agent and application thereof

Technical Field

The invention belongs to the technical field of biological RNA sample preservation, and particularly relates to a composition protective agent for preserving RNA in a liquid sample at normal temperature and an application method thereof.

Background

Prostate cancer is one of the most common cancers in men. Whether a patient has prostate cancer is based primarily on PSA, a cancer factor in the blood. However, due to the diagnostic accuracy as low as 30%, a considerable number of patients need to perform additional invasive biopsies, resulting in side effects such as bleeding and pain.

As a non-invasive method, diagnostic tests using urine are convenient for the patient, do not require invasive biopsy, and thus diagnose cancer without side effects. However, urine has low nucleic acid concentrations, with fewer prostate-derived components, and in particular, has low prostate-specific RNA levels, which in most cases cannot be detected immediately after extraction from a biological sample for a variety of reasons. Once isolated from a biological sample containing RNA, the RNA becomes very unstable and is very susceptible to degradation. The urine RNA preservative solution is usually required to be preserved under the condition of liquid nitrogen or-80 ℃, and the application of urine RNA detection is limited, so that the urine RNA preservative solution which can effectively prevent RNA degradation and does not influence the subsequent detection result is developed, is very important for the molecular biological detection of urine RNA, and has important significance for the development of the detection application of urine samples.

The current products aiming at RNA protection also have the following defects: 1. most patents aim at samples with high RNA content such as tissues, blood, swabs and the like, and aim at the scarce preservation solution of body fluid samples with low RNA content such as urine and the like. 2. The existing protective agent for preserving urine RNA, such as a Norgen urine preservation tube, needs to obtain RNA by extracting mixed liquid after preserving urine, the single volume of a sample extracted by a conventional extraction kit is 2-5ml, the extraction cost is very high, a urine RNA protective agent needs to be developed urgently, RNA in a precipitate can be extracted by a conventional Trizol method after the preserved urine sample is enriched and precipitated in a low-speed centrifugation mode, so that single extraction of dozens of ml urine samples can be easily realized, and the extraction cost is greatly reduced. 3. Reducing agents used in the existing protective agent for urine RNA preservation are beta-mercaptoethanol and dithiothreitol DTT, and the reducing agents are used for reducing disulfide bonds in proteins and can be used for preventing intra-molecular or intermolecular disulfide bonds of the proteins formed between cysteine in the proteins. However, beta-mercaptoethanol itself has only one mercapto group, and its effect is generally maintained for only 2-3 days. Therefore, the beta-mercaptoethanol is supplemented every 2 to 3 days in the experiment to maintain the effect of the beta-mercaptoethanol, and the beta-mercaptoethanol has volatility, strong irritation and toxicity. Dithiothreitol DTT has two sulfhydryl groups, is slightly more reductive than beta-mercaptoethanol, can maintain the action for 3-7 days, but often cannot reduce disulfide bonds embedded in the interior of a protein structure (inaccessible to a solvent), and the reduction of the disulfide bonds usually requires the protein to be firstly denatured (high-temperature heating or adding a denaturant such as 6M guanidine hydrochloride, 8M urea or 1% SDS). And DTT is easily oxidized by air, has poor stability, and needs to be preserved by freezing or treated in an inert gas to prolong its service life. Therefore, there is a need to find more stable and effective reducing agents. 4. The existing research aiming at the protective agent for urine RNA preservation mainly verifies that the total RNA content changes and mRNA amplification differences lack urine RNA fragment size distribution data and RNA-Seq verification data. And the size range of the RNA fragment and the RNA-Seq verification data are clear, so that the method plays an important role in the application field of RNA sequencing.

In addition, various types of urine samples can be encountered in clinical detection, the urine samples with very few clear, transparent and exfoliated cells and the samples with hematuria appear, so the storage difficulty of the RNA of the urine samples is increased due to the complexity of the urine samples, and the method is also embodied in the actual exploration process of the invention. The optimal chaotropic agent, settling agent, reducing agent and the like are used in different amounts under different sample states. A large number of samples need to be tested to obtain the protective agent suitable for various types of urine samples, and the protective effects of the protective agent on different samples are comprehensively balanced, so that the requirements of clinical practical application are met.

Disclosure of Invention

The invention aims to provide an RNA protective agent which can effectively maintain the stability of transported and stored liquid samples, particularly RNA in urine samples, and more importantly can be suitable for the storage of various urine samples. The urine sample preserved by the preserving fluid can be enriched and precipitated in a low-speed centrifugation mode, and then RNA in the precipitate is extracted by a conventional Trizol method, so that a convenient and fast mode is provided for downstream extraction, the extraction volume of the original urine sample is increased, the problem of difficulty in large-volume extraction of the urine sample is solved, and the extraction difficulty and the extraction cost are greatly reduced. The invention realizes the normal temperature storage and transportation of large-volume urine samples, and the extracted RNA can be well suitable for the subsequent RT-PCR and RNA-seq application. The excellent indexes of high concentration, high integrity, high stability after long-time storage and the like of the RNA stored in the storage solution are verified through various modes.

The invention is realized by adopting the following technical scheme:

an RNA protectant comprising the following components: chaotropic agents, precipitating agents, reducing agents, nuclease inhibitors; wherein:

the chaotropic agent includes: at least one of guanidinium isothiocyanate and guanidinium hydrochloride;

the reducing agent comprises: tris (2-carboxyethyl) phosphine hydrochloride;

the settling agent comprises: at least one of PEG and sulfosalicylic acid.

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

The concentration of the RNA protectant, tris (2-carboxyethyl) phosphine hydrochloride, in the protectant is 50-300mM, preferably 150-250mM, more preferably 180-220mM, and most preferably 200 mM.

The concentration of the RNA protective agent, PEG, in the protective agent is 5-30%, preferably 15-25%, further preferably 18-22%, and most preferably 20%; the concentration of sulfosalicylic acid in the protectant is 250mM-1M, preferably 300-800mM, more preferably 400-600mM, and most preferably 500 mM.

The RNA protective agent and the nuclease inhibitor comprise one or more of the following components: ethylenediaminetetraacetic acid (EDTA) and its salts, Ethylene Glycol Tetraacetic Acid (EGTA) and its salts, aurintricarboxylic acid (ATA), glyceraldehyde, NaF, formamide, vanadium-based ribonucleoside complexes, 8-hydroxyquinoline, bentonite, Sodium Dodecyl Sulfate (SDS), cysteine. . EDTA in the protective agent at a concentration of 5-100mM, preferably 30-60mM, further preferably 45-50mM, most preferably 50 mM;

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

the concentration of ATA in the protective agent is 0.5-10mM, preferably 2-8mM, further preferably 4-6mM, and most preferably 5 mM;

glyceraldehyde is present in the protective agent at a concentration of 50mM-200mM, preferably 60-150mM, further preferably 80-120mM, most preferably 100 mM;

the concentration of NaF in the protective agent is 1mg/ml-10mg/ml, preferably 3-6mg/ml, further preferably 4-5mg/ml, and most preferably 5 mg/ml;

the concentration of formamide in the protective agent is 10mg/ml-100mg/ml, preferably 30-60mg/ml, further preferably 40-50mg/ml, and most preferably 50 mg/ml;

the concentration of the vanadium-based nucleoside complex in the protective agent is 10mg/ml to 100mg/ml, preferably 30 mg/ml to 60mg/ml, further preferably 40 mg/ml to 50mg/ml, and most preferably 50 mg/ml;

the concentration of the 9-hydroxyquinoline in the protective agent is 0.5% -5%, preferably 1% -5%, further preferably 1% -2%, and most preferably 2%;

the concentration of the bentonite in the protective agent is 10mg/ml-100mg/ml, preferably 30-60mg/ml, further preferably 40-50mg/ml, and most preferably 50 mg/ml;

sodium Dodecyl Sulfate (SDS) in the protective agent at a concentration of 0.5% -5%, preferably 1% -3%, further preferably 1% -2%, most preferably 1%;

cysteine concentration in the protective agent is 5-25mM, preferably 10-25mg/ml, further preferably 15-20mg/ml, most preferably 20 mg/ml;

the RNA protective agent further comprises: a buffer and a solvent.

The RNA protective agent is used for protecting RNA,

the buffer comprises one or more of the following components: citric acid-sodium citrate buffer, Tris-HCl buffer, HEPES buffer.

The solvent is sterilized purified water.

The pH range of the RNA protective agent, namely the citric acid-sodium citrate buffer solution in the RNA protective agent is 4.0-6.5, preferably 4.5, and the concentration is 0.1M-0.2M, preferably 0.1M; the pH value of Tris-HCl buffer solution in the RNA protective agent ranges from 6.0 to 7.0, preferably 6.8, and the concentration is 0.1M to 0.2M, preferably 0.1M; HEPES buffer in RNA protective agent pH range 6.5-8.0, preferably pH7.0, concentration of 0.1-0.2M, preferably 0.1M.

The RNA protective agent further comprises: the lower alcohol, preferably the lower alcohol, comprises one or more of the following components: methanol, ethanol, isopropanol.

The content of the RNA protective agent, lower alcohol in the protective agent is 10-40%, preferably 10-30%, further preferably 15-25%, most preferably 20%;

the RNA protective agent further comprises: the surfactant, preferably the surfactant, comprises one or more of the following components: tween 20, Triton X-100, Nonidet P40 and Brij 35.

The content of the surface active agent in the RNA protective agent is 5-20%, preferably 5-15%, further preferably 8-12%, and most preferably 10%;

the second purpose of the invention is to provide the application of the RNA protective agent, which is used for preserving RNA in a liquid sample, especially RNA in a urine sample.

Further, the RNA protectant preserves urine at temperatures in the range of-20 ℃ to 37 ℃, preferably 4 ℃ to 37 ℃.

Further, urine is added to the RNA protectant after collection.

Furthermore, the volume ratio of the urine to the RNA protective agent is 3-5: 1, preferably 4: 1.

the invention has the beneficial effects.

Advantage 1: the test is carried out aiming at different types of urine samples in large-batch clinical practice, and the method can be suitable for storing various urine sample RNA.

Advantage 2: the precipitation aid used in the invention is used for enriching RNA by precipitating ribonucleoprotein, and the effect of adopting pure nucleic acid precipitation aid such as glycogen and LiCl reported in literature or patent is poor. The invention originally precipitates the ribonucleoprotein by a protein precipitation mode, further precipitates RNA in the ribonucleoprotein, and finds the precipitation aid particularly suitable for enriching the ribonucleoprotein in a urine sample and the optimal concentration proportion.

Advantage 3: RNA stored in the preservation solution can be enriched and precipitated to the bottom of the tube in a centrifugal mode, and after supernatant is removed, RNA in the precipitate is extracted, so that the problems that the extraction kit is limited and the extraction cost is increased due to the large volume when the urine sample has low RNA content and is extracted are solved. For example, the present invention can easily extract high concentration RNA by using a conventional Trizol method after 50ml urine is enriched and precipitated by using a 50ml centrifuge tube. While other urine preservation solutions such as urine collected by a urine preservation tube of Norgen can only extract 5ml or more of urine sample by using an extraction reagent specially aiming at extracting RNA in large volume, and the amount of RNA extracted by the urine sample is very limited.

Advantage 4: the reducing agent does not use dithiothreitol DTT and beta-mercaptoethanol, and adopts tris (2-carboxyethyl) phosphine hydrochloride (TCEP). TCEP is more reducing than both beta-mercaptoethanol and DTT, and its effect can be maintained for 2-3 weeks.

Advantage 5: the evaluation of the effect of the preservation solution on RNA preservation not only evaluates the RNA concentration and the application effect of RT-PCR, but also evaluates the integrity of RNA and the application effect of RNA-Seq, has more comprehensive verification data, and is beneficial to the application of downstream technology.

Advantage 6: the time limit of normal temperature storage can be as long as 20 days, and the stability is excellent; the problem of sample preservation and transportation after clinical sampling is solved.

Drawings

FIG. 1 is the analysis map of RNA Qsep100 fragment extracted from pure urine in 0 day in example 9;

FIG. 2 is the RNA Qsep100 fragment analysis map extracted from the urine preserved in the RNA preservation solution in example 9 for 10 days;

FIG. 3 is the analysis map of RNA Qsep100 fragment extracted from the urine preserved in the RNA preservation solution in example 9 for 20 days.

Detailed Description

The following examples are intended to further illustrate the invention without limiting it.

Example 1: synergistic effect of chaotropic agent and settling agent in the preserving fluid

Samples of normal human morning urine were collected and 16ml of each urine sample was added to 50ml centrifuge tubes/purified water centrifuge tubes containing the following sets of storage solutions. The volume ratio of the purified water or preservation solution to the urine is 1: 4. the room temperature is 23.6 ℃, and the solvent is sterilized pure water.

TABLE 1

And (3) enriching precipitates immediately after collecting urine of the group 1 purified water, adding an RZ reagent in a Tiangen total RNA extraction kit (DP419) into the precipitates, storing the precipitates in a refrigerator at the temperature of minus 80 ℃ for standby, and extracting total RNA of the urine after 5 days. Collecting urine of the purified water in the group 2, standing at normal temperature for 5 days, and enriching and precipitating with other preserving fluid groups to extract RNA. The 3 rd to 17 th groups of urine added with the preservation solution components are placed for 5 days at normal temperature and then centrifuged for 30min at 4000g to enrich and precipitate and extract RNA. Extraction reagent is Tiangen total RNA extraction kit (DP419) to extract urine total RNA, and ACTB and PSA gene mRNA is detected by using two-step RT-PCR, and the results are shown in Table 2:

TABLE 2

From the results of this example, the purified water group was left for 5 days at room temperature, and the urine was grown into bacteria, and the RNA was completely degraded. The solution of guanidine hydrochloride and guanidine isothiocyanate is added for clarification, and guanidine salt can inhibit the growth of bacteria. Group 13(4M guanidine hydrochloride, 20% PEG) and group 16(4M guanidine isothiocyanate, 20% PEG) were the best. When the guanidine salt concentration is too high (8M guanidine hydrochloride or 6M guanidine isothiocyanate), no flocculent precipitate exists at the tube bottom, so that RNA cannot be enriched, and when the guanidine salt concentration is too low (2M), the effect of inhibiting the RNase activity cannot be exerted, so that RNA degradation is caused. Through the combination of guanidine salt with proper concentration and PEG, the guanidine salt with proper concentration can inhibit the activity of RNase, meanwhile, ribonucleoprotein denaturation is not caused, RNA is dissolved and released, PEG can enhance the precipitation effect, and more RNA is obtained. This example provides a new enrichment concept: the RNA-containing ribonucleoprotein complex precipitate can be obtained under the condition of 4000g low-speed centrifugation by a way of enriching the ribonucleoprotein complex rather than a way of simply enriching nucleic acid.

However, the best preservation solution in this example, group 13(4M guanidine hydrochloride, 20% PEG) and group 16(4M guanidine isothiocyanate, 20% PEG), still have some differences from the results of the group 1 day 0 urine plus pure water, and the improvement of optimization will be shown in the following examples.

Example 2: nuclease inhibitor assay

Samples of normal human morning urine were collected and 16ml of each urine sample was added to 50ml centrifuge tubes/purified water centrifuge tubes containing the following sets of storage solutions. The volume ratio of the purified water or preservation solution to the urine is 1: 4. the room temperature is 23.4 ℃, and the solvent is sterilized pure water.

TABLE 3

Group number	Components
			1	Purifying water, enriching precipitate in 0 day
2	Purified water, standing at room temperature for 5 days
		3	4M guanidine hydrochloride, 20% PEG, 50mM EDTA
4	4M guanidinium isothiocyanate, 20% PEG, 50mM EDTA
		5	4M guanidine hydrochloride, 20% PEG, 50mM EGTA
6	4M guanidinium isothiocyanate, 20% PEG, 50mM EGTA
		7	4M guanidine hydrochloride, 20% PEG, 100mM glyceraldehyde
8	4M guanidinium isothiocyanate, 20% PEG, 100mM glyceraldehyde
		9	4M guanidine hydrochloride, 20% PEG, 5mM ATA
10	4M guanidinium isothiocyanate, 20% PEG, 5mM ATA
		11	4M guanidine hydrochloride, 20% PEG, 5mg/ml NaF
12	4M guanidinium isothiocyanate, 20% PEG, 5mg/ml NaF
		13	50mM EDTA
14	50mM EGTA
		15	100mM glyceraldehyde
16	5mM ATA
		17	5mg/ml NaF

And (3) enriching precipitates immediately after collecting urine of the group 1 purified water, adding an RZ reagent in a Tiangen total RNA extraction kit (DP419) into the precipitates, storing the precipitates in a refrigerator at the temperature of minus 80 ℃ for standby, and extracting total RNA of the urine after 5 days. Collecting urine of the purified water in the group 2, standing at normal temperature for 5 days, and enriching and precipitating with other preserving fluid groups to extract RNA. The 3 rd to 17 th groups of urine added with the preservation solution components are placed for 5 days at normal temperature and then centrifuged for 30min at 4000g to enrich and precipitate and extract RNA. Extraction reagent for total RNA extraction kit (DP419) from Tiangen total RNA, ACTB and PSA gene mRNA were detected using two-step RT-PCR, and the results are shown in Table 4:

TABLE 4

From the results of this example, the effect of adding only the nuclease inhibitor was poor. The addition of nuclease inhibitors based on the optimal group preservation solution of example 1 can suitably improve the RNA protection effect, and the effect of several nuclease inhibitors has no obvious difference.

Example 3: reductant test

On the basis of group 3(4M guanidine hydrochloride, 20% PEG, 50mM EDTA) and group 4(4M guanidine isothiocyanate, 20% PEG, 50mM EDTA) of example 2, it was expected to increase the RNA yield by adding a reducing agent.

Samples of normal human morning urine were collected and 16ml of each urine sample was added to 50ml centrifuge tubes/purified water centrifuge tubes containing the following sets of storage solutions. The volume ratio of the purified water or preservation solution to the urine is 1: 4. the room temperature is 24.1 ℃, and the solvent is sterilized pure water.

TABLE 5

Group number	Components
			1	Purifying water, enriching precipitate in 0 day
2	Purifying water, standing at room temperature for 5 days
		3	4M guanidine hydrochloride, 20% PEG, 50mM EDTA
4	4M guanidinium isothiocyanate, 20% PEG, 50mM EDTA
		5	4M guanidine hydrochloride, 20% PEG, 50mM EDTA, 8% beta-mercaptoethanol
6	4M guanidinium isothiocyanate, 20%PEG, 50mM EDTA, 8% beta-mercaptoethanol
		7	4M guanidine hydrochloride, 20% PEG, 50mM EDTA, 200mM DTT
8	4M guanidinium isothiocyanate, 20% PEG, 50mM EDTA, 200mM DTT
		9	4M guanidine hydrochloride, 20% PEG, 50mM EDTA, 200mM TCEP
10	4M guanidinium isothiocyanate, 20% PEG, 50mM EDTA, 200mM TCEP
		11	8% beta-mercaptoethanol
12	200mM DTT
		13	200mM TCEP

And (3) enriching precipitates immediately after collecting urine of the group 1 purified water, adding an RZ reagent in a Tiangen total RNA extraction kit (DP419) into the precipitates, storing the precipitates in a refrigerator at the temperature of minus 80 ℃ for standby, and extracting total RNA of the urine after 5 days. Collecting urine of the purified water in the group 2, standing at normal temperature for 5 days, and enriching and precipitating with other preserving fluid groups to extract RNA. The 3 rd to 13 th groups of urine added with the preservation solution components are placed for 5 days at normal temperature and then centrifuged for 30min at 4000g to enrich and precipitate and extract RNA. Extraction reagent for total RNA extraction kit (DP419) from Tiangen total RNA, ACTB and PSA gene mRNA were detected using two-step RT-PCR, with the results shown in Table 6:

TABLE 6

From the results of this example, it is found that the effect of adding only a reducing agent such as β -mercaptoethanol, DTT, TCEP, etc. is poor. The addition of the reducing agent based on the optimal group preservation solution of example 2 can improve the RNA protection effect, and the DTT and TCEP effects are better when the solution is preserved for 5 days at normal temperature. TCEP has good stability and solubility in aqueous solutions. The stability in acid and alkaline solution is good. Tests for stability and time-of-action of several reducing agents will be presented in the examples which follow.

Example 4: reductant stability and time-of-action test

Samples of normal human morning urine were collected and 16ml of each urine was added to the following 50ml tubes of each storage solution/purified water set. The volume ratio of purified water or preservative fluid/purified water volume to urine is 1: 4. the room temperature is 23.8 ℃, and the solvent is sterilized pure water.

And (3) enriching precipitates immediately after collecting urine of the group 1 purified water, adding an RZ reagent in a Tiangen total RNA extraction kit (DP419) into the precipitates, storing the precipitates in a refrigerator at the temperature of minus 80 ℃ for standby, and extracting total RNA of the urine after 5 days. And (3) placing the sample added with purified water or preservation solution in groups 2-21 at normal temperature for 3 days, 6 days, 10 days, 20 days and 30 days, and then centrifuging for 30min by 4000g to enrich and precipitate and extract RNA. Extraction reagent for total RNA extraction kit (DP419) from Tiangen total RNA, ACTB and PSA gene mRNA were detected using two-step RT-PCR, with the results shown in Table 7:

TABLE 7

From the results of this example, the effect of β -mercaptoethanol can only be maintained for 3 days, and the effect is significantly reduced when stored for 6 days; the DTT effect can be maintained for 6 days, and the effect is obviously reduced when the DTT is stored for 10 days; the effect of TCEP is stable after the TCEP is stored for 20 days, and the effect is slightly reduced after the TCEP is stored for 30 days. Therefore, TCEP is selected as the reducing agent in the preserving fluid of the invention.

Example 5: different urine sample testing

The following components are prepared:

the solvent is sterilized pure water.

TABLE 8

	Components
		Contrast agent	Purified water
Preservative solution reagent	4M guanidinium isothiocyanate, 20% PEG, 50mM EDTA, 200mM TCEP

The method comprises the following steps of collecting 8 normal human urine samples (NM group), 8 prostate cancer urine samples (PCA group), 8 bladder cancer urine samples (BC group) and 8 bladder cancer hematuria samples (BCB group), taking 16ml after collection, respectively adding the 16ml into a centrifugal tube containing a contrast reagent and a preservation solution reagent, wherein the volume ratio of purified water/preservation solution to urine is 1: 4. room temperature 23.8 ℃.

Adding into urine of contrast reagent, enriching precipitate immediately after 0 day, adding RZ reagent in Tiangen total RNA extraction kit (DP419) into precipitate, storing in-80 deg.C refrigerator for use, and extracting urine total RNA after 5 days. The urine added with the preservation solution reagent is preserved at room temperature for 5 days, then total RNA of the urine is extracted by using a total RNA extraction kit (DP419), PSA gene mRNA is detected by using two-step RT-PCR, and the results are shown in Table 9:

TABLE 9

The result shows that the sedimentation enrichment effect of the urine sample with more exfoliated cells after centrifugation is obvious, and the result difference of the urine sample with more exfoliated cells and the result difference of the urine sample with the 0-day control group is smaller. For urine samples with few exfoliated cells, there was almost no visible precipitate after centrifugation, which was significantly different from the results of the day 0 control group. The formula of the preservation solution is prompted to be optimized according to different urine sample types, so that the result of the sample with few exfoliated cells can reach the level of a control group, and the clinical requirements of various types of samples are met. The optimization of which will be shown in the subsequent embodiments.

Example 6: settling aid screening test

Based on the results of example 5, this example is expected to improve the effect of sediment enrichment by screening various settling agents to meet the requirement of samples with fewer exfoliated cells.

2 normal morning urine samples (with more exfoliated cells) were collected, and the sample was numbered A, B; random urine samples (few exfoliated cells) from 2 normal persons were numbered C, D. After collection, 16ml of each urine sample was added to 50ml centrifuge tubes/purified water centrifuge tubes containing the following respective sets of purified water or storage solutions. The volume ratio of the purified water or preservation solution to the urine is 1: 4. room temperature 24.6 ℃.

Watch 10

And (3) enriching precipitates immediately after collecting urine of the group 1 purified water, adding an RZ reagent in a Tiangen total RNA extraction kit (DP419) into the precipitates, storing the precipitates in a refrigerator at the temperature of minus 80 ℃ for standby, and extracting total RNA of the urine after 5 days. Collecting urine of the purified water in the group 2, standing at normal temperature for 5 days, and enriching and precipitating with other preserving fluid groups to extract RNA. The 3 rd to 15 th groups of urine added with the preservation solution components are placed for 5 days at normal temperature and then centrifuged for 30min at 4000g to enrich and precipitate and extract RNA. The extraction reagent is a Tiangen total RNA extraction kit (DP419) for extracting urine total RNA, PSA gene mRNA is detected by using two-step RT-PCR, and the results are shown in Table 11:

TABLE 11

The results show that sulfosalicylic acid and PEG helped the sediment better, and group 13(4M guanidinium isothiocyanate, 50mM EDTA, 200mM TCEP, 20% PEG, 500mM sulfosalicylic acid, 20% isopropanol) was the best for the two samples with fewer exfoliated cells. The effect of the settling aid capable of precipitating protein is better than that of the settling aid only capable of precipitating nucleic acid. The protective agent of the invention proved to work best in a manner that precipitates ribonucleoproteins.

Example 7: preservative fluid optimization test

Based on example 6 group 13(4M guanidinium isothiocyanate, 50mM EDTA, 200mM TCEP, 20% PEG, 500mM sulfosalicylic acid, 20% isopropanol), this example is based on the fact that surfactants can reduce adsorption of nucleic acid protein molecules on the tube wall, buffers can maintain stability of the solution, and tests of surfactants and buffers are increased for samples with few exfoliated cells.

2 normal morning urine samples (with more exfoliated cells) were collected, and the sample was numbered A, B; random urine samples (few exfoliated cells) from 2 normal persons were numbered C, D. After collection, 16ml of each urine sample was added to 50ml centrifuge tubes/purified water centrifuge tubes containing the following respective sets of purified water or storage solutions. The volume ratio of the purified water or preservation solution to the urine is 1: 4. room temperature 24.5 ℃.

TABLE 12

And (3) enriching precipitates immediately after collecting urine of the group 1 purified water, adding an RZ reagent in a Tiangen total RNA extraction kit (DP419) into the precipitates, storing the precipitates in a refrigerator at the temperature of minus 80 ℃ for standby, and extracting total RNA of the urine after 5 days. Collecting urine of the purified water in the group 2, standing at normal temperature for 5 days, and enriching and precipitating with other preserving fluid groups to extract RNA. The 3 rd to 10 th groups of urine added with the preservation solution components are placed for 5 days at normal temperature and then centrifuged for 30min by 4000g to enrich and precipitate and extract RNA. The extraction reagent is a Tiangen total RNA extraction kit (DP419) for extracting urine total RNA, PSA gene mRNA is detected by using two-step RT-PCR, and the results are shown in Table 13:

watch 13

The results show that the results for each surfactant are slightly better than the non-surfactant group, and that the buffered environment works better under acidic and neutral conditions.

Example 8: preservation temperature range test of preservation solution for urine preservation

Preparing an RNA preservation solution for the urine sample, and controlling the contents of the components as follows: preparing an RNA preservation solution for the urine sample, and controlling the contents of the components as follows: 4M guanidinium isothiocyanate, 50mM EDTA, 200mM TCEP, 20% PEG, 500mM sulfosalicylic acid, 20% isopropanol, 10% Tween 20, 0.1M citric acid-sodium citrate buffer (pH 4.5). And respectively injecting 2mL of the prepared preservation solution into a vacuum urine collection tube for collecting 8mL of urine, fully reversing and uniformly mixing for 8-10 times, and preparing 12 tubes. The obtained extract was stored at-20 deg.C, 4 deg.C, room temperature, and 37 deg.C for 20 days, and each group was set to have 3 replicates. In addition, 2 tubes of 8ml of pure urine were prepared, 2ml of sterilized purified water was added, and after 0 day of precipitation, total RNA in urine was extracted by Trizol method and stored at-80 ℃. The samples of each group added with RNA preservation solution are collected at 10 and 20 days respectively, and urine total RNA is extracted by using a Trizol method and preserved at-80 ℃. The extraction reagent is a Tiangen total RNA extraction kit (DP419) for extracting urine total RNA, PSA gene mRNA is detected by using a two-step RT-PCR method, and the ct value of the detection result is shown in the following table:

TABLE 14

Serial number	Day 0 control group	-20℃	4℃	At 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

The result shows that the effect of the preservation temperature of the preservation solution for preserving the urine in the range of-20 ℃ to 37 ℃ is not obviously different from that of a 0-day control group.

Example 9: RNA integrity test and RNA-seq result evaluation

Preparing an RNA preservation solution for the urine sample, and controlling the contents of the components as follows: preparing an RNA preservation solution for the urine sample, and controlling the contents of the components as follows: 4M guanidinium isothiocyanate, 50mM EDTA, 200mM TCEP, 20% PEG, 500mM sulfosalicylic acid, 20% isopropanol, 10% Tween 20, 0.1M citric acid-sodium citrate buffer (pH 4.5). And respectively injecting 2mL of the prepared preservation solution into a vacuum urine collection tube for collecting 8mL of urine, fully reversing and uniformly mixing for 8-10 times, and preparing 2 tubes. Storing at 25 + -5 deg.C for 10 and 20 days. In addition, 1 tube of 8ml of pure urine and 2ml of sterilized purified water were prepared, and after collecting the precipitate for 0 day, total RNA in the urine was extracted by Trizol method and stored at-80 ℃. The samples of each group added with RNA preservation solution are collected at 10 and 20 days respectively, and urine total RNA is extracted by using a Trizol method and preserved at-80 ℃. All RNAs were analyzed for RNA fragment size using a Qsep100 fragment analyzer, and the results are shown in FIGS. 1, 2, and 3; the extracted RNA was subjected to library sequencing, and the results are shown in table 15:

watch 15

Example 9 the size of the extracted RNA fragments was tested and the results showed that the RNA fragment distribution after 10 and 20 days of storage of the storage solution was almost identical to that of 0 day pure urine. The RNA-Seq results were not significantly different from the results of the 0-day pure urine control group. The preservation solution can ensure the integrity of RNA, and the result of RNA sequencing is real and reliable.

Example 10: comparative testing with Norgen urine storage tubes

The urine sample RNA preservative fluid is prepared to be used as a preservative fluid testing group (DDTN group) of the invention, and the contents of the components are controlled as follows: 4M guanidinium isothiocyanate, 50mM EDTA, 200mM TCEP, 20% PEG, 500mM sulfosalicylic acid, 20% isopropanol, 10% Tween 20, 0.1M citric acid-sodium citrate buffer (pH 4.5). Norgen Urine storage tubes were purchased from the Urine Collection and Preservation Tube (Cat.18120) of Norgen Biotek Corp. Set 3 groups: after 8ml of urine is respectively added into a DDTN group, a Norgen precipitation group and a Norgen mixed liquor group, the DDTN group is placed for 20 days, the DDTN group is centrifuged for 20min according to a sample pretreatment mode of the preservation solution, the precipitate is reserved for extracting urine total RNA by using a total root RNA extraction kit (DP419), the Norgen precipitation group is centrifuged for 20min according to a sample pretreatment mode of 6000g, the precipitate is reserved for extracting urine total RNA by using a total root RNA extraction kit (DP419), and the Norgen mixed liquor group is directly extracted by using the total 8ml of mixed liquor in the preservation tube according to the recommendation (the extraction kit is Zymo Quick-cfRNA)^TMSerum&Plasma Kit (R1059)), and RT-PCR was performed on the extracted RNA, setting 3 tube repeats, with the following results:

TABLE 16

From the test results, the extraction effect of the DDTN precipitation group is the best, the extraction mode of the Norgen urine storage tube after enrichment precipitation has poor effect, RNA can be obtained only by extracting large-volume mixed liquid in a mode recommended by the storage liquid, and the cost of the extraction kit used in the mode is more than 10 times that of the total RNA extraction kit.

Removing rRNA from the RNA extracted from the 3 groups, and performing library construction and sequencing, wherein the rRNA removing reagent is Hieff

One-Step rRNA Removal Kit (Cat No.12258), results are shown in the following table:

TABLE 16

The RNA-Seq result shows that the map rate of the Norgen precipitation group and the Norgen mixed solution group is obviously lower than that of the DDTN group, which indicates that the effect of storing the human RNA in urine by the DDTN storage solution is better than that of the Norgen storage tube; biological information analysis shows that most of UNMAPPED READS (data not compared with the human transcriptome) is compared with the microbial genome, so that a great amount of microbial pollution exists in the Norgen precipitation group and the Norgen mixed liquid group, and the bacteriostatic effect of the UNMAPPED READS in the urine sample preservation is not as good as that of the DDTN group.

Claims

1. An RNA protective agent, which is characterized by comprising the following components: chaotropic agents, precipitating agents, reducing agents, nuclease inhibitors; wherein:

the reducing agent comprises: tris (2-carboxyethyl) phosphine hydrochloride;

the settling agent comprises: at least one of PEG and sulfosalicylic acid.

2. The RNA protectant according to claim 1, wherein the concentration of the chaotropic agent in the protectant is 3-5M, preferably 3.5-4.5M, further preferably 3.8-4.2M, most preferably 4M.

3. The RNA protectant according to claim 1, wherein the concentration of tris (2-carboxyethyl) phosphine hydrochloride in the protectant is 50-300mM, preferably 150-250mM, more preferably 180-220mM, most preferably 200 mM.

4. The RNA protectant according to claim 1, wherein the concentration of PEG in the protectant is 5-30%, preferably 15-25%, further preferably 18-22%, most preferably 20%; the concentration of sulfosalicylic acid in the protectant is 250mM-1M, preferably 300-800mM, more preferably 400-600mM, and most preferably 500 mM.

5. The RNA protecting agent according to claim 1,

the nuclease inhibitor comprises one or more of the following components: ethylenediaminetetraacetic acid and salts thereof, ethylene glycol tetraacetic acid and salts thereof, aurin tricarboxylic acid, glyceraldehyde, NaF, formamide, vanadium-based nucleoside complex, 8-hydroxyquinoline, bentonite, sodium dodecyl sulfate, and cysteine.

6. The RNA protectant according to claim 5,

the concentration of the ethylene diamine tetraacetic acid and salts thereof in the protective agent is 5-100mM, preferably 30-60mM, further preferably 45-50mM, and most preferably 50 mM;

the concentration of the ethylene glycol tetraacetic acid and the salt thereof in the protective agent is 5mM-100mM, preferably 30-60mM, further preferably 45-50mM, and most preferably 50 mM;

aurintricarboxylic acid in the protective agent at a concentration of 0.5-10mM, preferably 2-8mM, more preferably 4-6mM, most preferably 5 mM;

the concentration of 8-hydroxyquinoline in the protective agent is 0.5% -5%, preferably 1% -5%, further preferably 1% -2%, and most preferably 2%;

the concentration of the sodium dodecyl sulfate in the protective agent is 0.5% -5%, preferably 1% -3%, further preferably 1% -2%, and most preferably 1%;

the concentration of cysteine in the protective agent is 5-25mM, preferably 10-25mg/ml, more preferably 15-20mg/ml, and most preferably 20 mg/ml.

7. The RNA protective agent of claim 1, further comprising: a buffer and a solvent.

8. The RNA protectant according to claim 7,

the buffer comprises one or more of the following components: citric acid-sodium citrate buffer solution, Tris-HCl buffer solution and HEPES buffer solution;

the solvent is sterilized purified water.

9. The RNA protectant according to claim 8, wherein

The pH range of the citric acid-sodium citrate buffer solution in the RNA protective agent is 4.0-6.5, preferably pH4.5, and the concentration is 0.1M-0.2M, preferably 0.1M; the pH value of Tris-HCl buffer solution in the RNA protective agent ranges from 6.0 to 7.0, preferably 6.8, and the concentration is 0.1M to 0.2M, preferably 0.1M; HEPES buffer in RNA protective agent pH range 6.5-8.0, preferably pH7.0, concentration of 0.1-0.2M, preferably 0.1M.

10. The RNA protective agent of claim 1, further comprising: the lower alcohol, preferably the lower alcohol, comprises one or more of the following components: methanol, ethanol, isopropanol.

11. The RNA protectant according to claim 10, wherein the lower alcohol is present in the protectant in an amount of 10-40%, preferably 10-30%, further preferably 15-25%, most preferably 20%.

12. The RNA protective agent of claim 1, further comprising: the surfactant, preferably the surfactant, comprises one or more of the following components: tween 20, Triton X-100, Nonidet P40 and Brij 35.

13. The RNA protectant according to claim 12, wherein the surfactant is present in the protectant in an amount of 5-20%, preferably 5-15%, further preferably 8-12%, most preferably 10%.

14. Use of an RNA protectant according to any of claims 1-13 for preserving RNA in a liquid sample, in particular in a urine sample.

15. Use according to claim 14, characterized in that the RNA protective agent is used at a temperature in the range-20 to 37 ℃, preferably 4 to 37 ℃.

16. The use of claim 14, wherein urine is added to said RNA protective agent after collection.

17. The use of claim 16, wherein the volume ratio of urine to RNA protectant is 3-5: 1, preferably 4: 1.