阅读说明：本技术 一种rna保护剂及其应用 (RNA protective agent and application thereof ) 是由 王进产 于 2021-03-22 设计创作，主要内容包括：本发明公开了一种RNA保护剂,该保护剂中的β-巯基乙醇、硫脲、乙二胺四乙酸二钠与盐酸胍共同作用可对RNase产生强烈的抑制作用,有效减弱核糖核酸酶对RNA的分解,有助于延长保持样本中RNA稳定性的时间。本发明还公开了一种RNA保护剂在保持样本中RNA稳定性的应用。经本发明得到的RNA保护剂保存的样本,其所含的RNA可用于所有关于RNA的后续实验,避免了液态保存RNA样本反复冻融对样本造成的损伤。本发明得到的RNA保护剂不影响RNA的完整性和活性,对RNA的保存、反复使用和长途运输具有重要的应用价值。(The invention discloses an RNA protective agent, wherein beta-mercaptoethanol, thiourea, ethylene diamine tetraacetic acid disodium and guanidine hydrochloride in the protective agent act together to generate strong inhibition effect on RNase, effectively weaken the decomposition of ribonuclease on RNA and contribute to prolonging the time for keeping the RNA stability in a sample. The invention also discloses application of the RNA protective agent in maintaining the stability of RNA in a sample. The RNA contained in the sample preserved by the RNA protectant obtained by the invention can be used for all subsequent experiments related to RNA, and the damage to the sample caused by repeated freeze thawing of the RNA sample preserved in a liquid state is avoided. The RNA protective agent obtained by the invention does not influence the integrity and activity of RNA, and has important application value for the storage, the repeated use and the long-distance transportation of RNA.)

1. An RNA protective agent, which is characterized by comprising the following components: guanidine hydrochloride, sodium citrate, beta-mercaptoethanol, thiourea, disodium ethylene diamine tetraacetate, sodium dodecyl sarcosinate, trehalose, Tween 80, Proclin300 and a solvent.

2. The RNA protective agent of claim 1, wherein the content of each component is: 1-6 mol/L guanidine hydrochloride, 22-28 mmol/L sodium citrate, 0.05-0.3mol/L beta-mercaptoethanol, 45-54 mmol/L thiourea, 8-12 mmol/L disodium ethylenediamine tetraacetate, 0.2-0.8 wt% sodium dodecyl sarcosinate, 0.6-1.2 wt% trehalose, 0.02-0.1% Tween 80(v/v) and 0.05-0.5% Proclin300 (v/v).

3. The RNA protective agent of claim 2, wherein the content of each component is: 4mol/L guanidine hydrochloride, 25mmol/L sodium citrate, 0.1mol/L beta-mercaptoethanol, 50mmol/L thiourea, 10mmol/L disodium ethylene diamine tetraacetate, 0.5 wt% sodium dodecyl sarcosinate, 1 wt% trehalose, 0.05% tween 80(v/v) and 0.1% Proclin300 (v/v).

4. The RNA protectant of claim 1, wherein the solvent is water.

5. Use of an RNA protectant according to any of claims 1-4 for maintaining RNA stability in a sample.

6. The use of claim 5, wherein the sample is one of a virus, a cell, a bacterium, a tissue, a secretion, a swab.

7. The use according to claim 6, wherein the sample is stored at a temperature of-80 ℃ to 37 ℃.

8. The use of claim 6, wherein said swab is selected from the group consisting of a blood swab, a nasal swab, and an oral swab.

9. The use of claim 5, wherein the protective agent is used by immersing the RNA-containing sample in the RNA protective agent.

Technical Field

The invention belongs to the technical field of biology, and relates to an RNA protective agent and application thereof.

Background

Ribonucleic acid (RNA) is a genetic information carrier existing in biological cells and partial viruses and viroids. A nucleic acid having at least several tens of ribonucleotides connected by phosphodiester bonds is named because of the ribose. Each RNA molecule consists of a long chain of nucleotide units, each nucleotide nitrogen source containing a nitrogenous base, a ribose sugar and a phosphate group. RNA is one of the main components of cells, participates in various functional activities of the cells, is an important research object of life-related disciplines such as biology, medicine and pharmacy, and RNA detection is an important technology of molecular biology research, including biochips, gene expression matrix analysis, quantitative RT-PCR detection, molecular diagnosis and the like. RNA detection has the following advantages in clinical diagnosis of pathogen infection: 1. the target is RNA, the number of amplification revealing templates is high, and the detection sensitivity is high; 2. after the pathogen dies, the RNA degradation speed is high, and the detection result can effectively distinguish whether the infection is transient infection or not, thereby being beneficial to clinical judgment and treatment scheme selection and avoiding ineffective treatment.

However, in most cases, for various reasons, RNA cannot be detected immediately after it has been extracted from a biological sample. Once isolated from a biological sample containing RNA, the RNA becomes very unstable and is very susceptible to degradation. External physical factors such as temperature, humidity, ultraviolet light that cause RNA degradation; chemical factors such as pH, hydrolysis, oxidation, etc.; biological factors such as enzymolysis, microbial infection and the like. And the RNA is a single-stranded structure, and is very easy to degrade in the extraction process or the storage process.

Therefore, the extraction and preservation process of RNA requires strict treatment to prevent specific or non-specific degradation of RNA, so as to facilitate subsequent research and use. The quality and integrity of RNA in the preservation process have a great influence on the accuracy and reliability of the detection result. RNA hydrolysis by the widely-existing and extremely-stable ribonuclease is strong, and RNA degradation by many ribonucleases does not need any auxiliary factor, so that purification, storage and use of RNA are greatly hindered. For tissue samples, the current method for preserving samples is mainly direct cryopreservation after sample collection, such as liquid nitrogen preservation which is commonly used. However, the method has great limitations, namely, the low-temperature preservation cost is high; secondly, the preservation equipment has higher requirements (such as a liquid nitrogen tank), if a freshly collected sample cannot be immediately placed in liquid nitrogen, RNA molecules may be degraded, and other influence factors may also change the state of RNA, thereby bringing operation errors to subsequent experiments. Therefore, it is very important to provide an RNA protective agent that can effectively prevent RNA degradation without affecting the subsequent detection results for the molecular biological detection of RNA, and it is also a technical problem that needs to be solved urgently for RNA preservation at present.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide an RNA protective agent which can effectively maintain the stability of RNA in a transport and storage sample.

The second object of the present invention is the use of an RNA protectant for maintaining RNA stability in a sample.

One of the purposes of the invention is realized by adopting the following technical scheme:

an RNA protectant comprising the following components: guanidine hydrochloride, sodium citrate, beta-mercaptoethanol, thiourea, disodium ethylene diamine tetraacetate, sodium dodecyl sarcosinate, trehalose, Tween 80, Proclin300 and a solvent.

Further, the content of each component is as follows: 1-6 mol/L guanidine hydrochloride, 22-28 mmol/L sodium citrate, 0.05-0.3mol/L beta-mercaptoethanol, 45-54 mmol/L thiourea, 8-12 mmol/L disodium ethylenediamine tetraacetate, 0.2-0.8 wt% sodium dodecyl sarcosinate, 0.6-1.2 wt% trehalose, 0.02-0.1% Tween 80(v/v) and 0.05-0.5% Proclin300 (v/v).

Further, the content of each component is as follows: 4mol/L guanidine hydrochloride, 25mmol/L sodium citrate, 0.1mol/L beta-mercaptoethanol, 50mmol/L thiourea, 10mmol/L disodium ethylene diamine tetraacetate, 0.5 wt% sodium dodecyl sarcosinate, 1 wt% trehalose, 0.05% tween 80(v/v) and 0.1% Proclin300 (v/v).

Further, the solvent is water.

The second purpose of the invention is realized by adopting the following technical scheme:

use of an RNA protective agent for preserving the stability of RNA in a sample.

Further, the sample is one of a virus, a cell, a bacterium, a tissue, a secretion, and a swab.

Further, the storage temperature of the sample is-80 ℃ to 37 ℃.

Further, the swab is selected from one of a blood swab, a nasal swab and an oral swab.

Further, the protective agent is used by immersing the RNA-containing sample in the RNA protective agent.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention provides an RNA protective agent, wherein guanidine hydrochloride is used as chaotropic salt and has an inhibiting effect on RNA enzyme; beta-mercaptoethanol and thiourea are used as antioxidants to reduce the biological activity of enzymes, disodium ethylene diamine tetraacetate can chelate divalent metal ions so as to inhibit the activity of ribonuclease, and the combined action of the beta-mercaptoethanol, the thiourea, the disodium ethylene diamine tetraacetate and guanidine hydrochloride can generate strong inhibition effect on the RNase, effectively weaken the decomposition of the ribonuclease on RNA and contribute to prolonging the time for keeping the RNA stability.

2. The invention also provides application of the RNA protective agent in keeping the stability of RNA in a sample, and the RNA protective agent can prolong the storage time of the RNA-containing sample at different temperatures. The RNA protective agent can be used for storing RNA samples for 3 days at 37 ℃, 7 days at 18-25 ℃, 4 weeks at 2-8 ℃, and long-term storage at-20 ℃ or-80 ℃. Although the sample preserved by the method of repeated freeze thawing still enables the RNA to have high biological activity, the RNA contained in the sample preserved by the RNA protectant obtained by the invention can be used for all subsequent experiments about the RNA, and the damage to the sample caused by the repeated freeze thawing of the liquid preserved RNA sample is avoided. The RNA protective agent obtained by the invention does not influence the integrity and activity of RNA, and has important application value for the storage, the repeated use and the long-distance transportation of RNA.

Detailed Description

The present invention is further described below with reference to specific embodiments, and it should be noted that, without conflict, any combination between the embodiments or technical features described below may form a new embodiment.

Example 1

An RNA protective agent comprising the following components:

4mol/L guanidine hydrochloride, 25mmol/L sodium citrate, 0.1mol/L beta-mercaptoethanol, 50mmol/L thiourea, 10mmol/L disodium ethylene diamine tetraacetate, 0.5 wt% sodium dodecyl sarcosinate, 1 wt% trehalose, 0.05% tween 80(v/v), 0.1% Proclin300(v/v), and the solvent is water.

The RNA protective agent obtained after the preparation is clear and transparent liquid.

The resulting RNA protective agent is used to protect the stability of RNA in viruses: collecting 10 of the extract stored at-70 deg.C^4.0TCID₅₀Each of the swine fever virus solution and the blue ear virus solution is 24, and each of the three solutions is 0.5 mL. The experiment was divided into 8 groups of 3 in each group at different storage temperatures and times, wherein each group was stored at the temperature described in Table 1 for a period of time after adding 0.5mL of the RNA protective agent obtained in example 1 to each group.

Example 2

An RNA protective agent comprising the following components:

guanidine hydrochloride 1mol/L, sodium citrate 22mmol/L, beta-mercaptoethanol 0.05mol/L, thiourea 45mmol/L, disodium ethylene diamine tetraacetate 8mmol/L, sodium dodecyl sarcosinate 0.2 wt%, trehalose 0.6 wt%, Tween 80(v/v) 0.02%, Proclin300(v/v) 0.05%, and water as solvent.

The RNA protective agent obtained after the preparation is clear and transparent liquid.

The resulting RNA protective agent is used to protect the stability of RNA in cells: performing conventional cell culture on a 293T cell sample to be preserved for 2-3 days, sucking a culture solution supernatant, transferring the culture solution supernatant into a centrifuge tube, centrifuging at 1500r/min for 10min, collecting cells, and discarding the supernatant; the collected cells were then washed with ice-bath buffer PBS and suspended in a small amount of buffer. The RNA protective agent obtained in example 2 was added in an amount of 5 to 10 times the volume of the RNA protective agent, mixed well, and stored in a collection tube. The experiment was divided into 8 groups according to different storage temperature-time, wherein 3 samples per group were subjected to storage tests under temperature-time conditions as described in table 1.

Example 3

An RNA protective agent comprising the following components:

guanidine hydrochloride 6mol/L, sodium citrate 28mmol/L, beta-mercaptoethanol 0.3mol/L, thiourea 54mmol/L, disodium ethylene diamine tetraacetate 12mmol/L, sodium dodecyl sarcosinate 0.8 wt%, trehalose 1.2 wt%, Tween 80(v/v) 0.1%, Proclin300(v/v) 5%, and water as solvent.

The RNA protective agent obtained after the preparation is clear and transparent liquid.

The resulting RNA protective agent is used to protect the stability of RNA in bacteria: transferring a streptococcus thermophilus Cas9 sample to be stored into a centrifuge tube, centrifuging at 1500r/min for 10min, collecting bacteria, and discarding supernatant; the collected bacteria were then washed with ice-bath buffer PBS and suspended in a small amount of buffer. The RNA protective agent obtained in example 3 was added in an amount of 5 to 10 times the volume of the RNA protective agent, mixed well, and stored in a collection tube. The experiment was divided into 8 groups according to different storage temperature-time, wherein 3 samples per group were subjected to storage tests under temperature-time conditions as described in table 1.

Example 4

Example 4 differs from example 1 in that: the protected object was changed to a histopathological material, a fresh liver histopathological material containing a blue-ear disease virus was collected, fragments each side of which was not more than 0.5cm were cut with scissors, the fragments of the tissues were completely immersed in a collection tube containing 2 to 3 times the volume of the RNA protectant obtained in the present invention, and subjected to a preservation test under the temperature-time conditions as described in Table 1. The rest is the same as in example 1.

Example 5

Example 5 differs from example 1 in that: the protected subjects were changed to secretions, and fresh saliva containing the blue-ear disease virus was taken and added directly to a container containing an equal volume of the RNA protective agent of the present invention and subjected to the storage test under temperature-time conditions as described in table 1. The rest is the same as in example 1.

Example 6

Example 6 differs from example 1 in that: the subjects were changed to swabs, and fresh blood swabs containing the blue-ear disease virus were collected, immediately immersed in a container containing an appropriate amount of the RNA protective agent of the present invention, and subjected to preservation test under temperature-time conditions as described in table 1. The rest is the same as in example 1.

Comparative example 1

Comparative example 1 differs from example 1 in that an equal amount of PBS is added instead of the RNA protectant as a negative control, and the rest is the same as example 1.

Comparative example 2

Comparative example 2 is different from example 1 in that thiourea and disodium ethylenediaminetetraacetate are not added to the RNA protectant, and the rest is the same as example 1.

Comparative example 3

Comparative example 3 is different from example 1 in that beta-mercaptoethanol and disodium ethylenediaminetetraacetate are not added to the RNA protectant, and the rest is the same as example 1.

Comparative example 4

Comparative example 4 is different from example 1 in that ethylenediaminetetraacetic acid is replaced with ethylenediaminetetraacetic acid, guanidine isothiocyanate is replaced with guanidine hydrochloride, and the rest is the same as example 1.

Experimental example 1

In order to examine the protective effect of the RNA protective agent obtained by the present invention on RNA-containing samples, samples stored in examples 1 to 6 and comparative examples 1 to 4 were stored at temperatures and times shown in the following table, RNA is extracted by using commercial kits (Loyang Aisen Biotechnology Co., Ltd., product No. AS201), fluorescent quantitative RT-PCR detection is carried out by using a porcine reproductive and respiratory syndrome virus general type (PRRSV-U) nucleic acid detection kit (a fluorescent-PCR method, Guangzhou Wei Berxin Biotechnology Co., Ltd., product No. SD-R-0211), the RNA stability in the sample after storage is presented by Ct value, the RNA degradation is more serious when the Ct value change rate (Ct value measured after storing corresponding temperature-time/Ct value stored for 0 day at corresponding temperature-1) x 100%) is higher, and the average value of the Ct value change rate of the sample stored under each temperature-time condition is shown in the table.

The RNA protective agent is used for protecting the stability of RNA in classical swine fever viruses and blue ear viruses, and the specific performance is shown in Table 1:

TABLE 1

The RNA protective agent is used for protecting the stability of RNA in 293T cells, and the specific performance is shown in Table 2:

TABLE 2

The RNA protective agent is used for protecting the stability of RNA in the streptococcus thermophilus Cas9, and the specific performance is shown in Table 3:

TABLE 3

The RNA protective agent is used for protecting liver tissue pathological material containing the blue-ear disease virus, and the specific performance is shown in Table 4:

TABLE 4

The RNA protective agent is used for protecting liver tissue pathological material containing the blue-ear disease virus, and the specific performance is shown in Table 4:

TABLE 5

The RNA protective agent is used for protecting liver tissue pathological material containing the blue-ear disease virus, and the specific performance is shown in Table 4:

TABLE 6

The RNA protective agent obtained in comparative examples 1 to 4 of the invention is used for protecting the stability of RNA in classical swine fever virus, and the specific performance is shown in Table 7:

TABLE 7

Temperature/time	Comparative example 1	Comparative example 2	Comparative example 3	Comparative example 4
					37 ℃/3 days	15.38％	10.51％	11.13％	7.56％
37 ℃/5 days	20.02％	21.35％	20.38％	18.96％
					25 ℃/3 days	6.93％	5.68％	5.23％	3.09％
25 ℃/7 days	9.21％	4.23％	4.68％	3.85％
					4 deg.C/2 weeks	10.01％	5.98％	5.21％	2.63％
4 deg.C/4 weeks	13.49％	10.59％	9.38％	5.26％
					20 ℃ per long term	0.67％	0.66％	0.60％	0.65％
80 ℃ per long term	0.59％	0.55％	0.51％	0.61％

As is clear from Table 1, the addition of the RNA protectant effectively prolongs the storage time of the RNA sample, and the RNA protectant of the present invention enables the RNA-containing sample to be stored at 37 ℃ for 3 days, 25 ℃ for 7 days, and 8 ℃ for 4 weeks, and can be stored at-20 ℃ for a long period of time. Comparative examples 1 to 4 showed no significant difference in storage at-20 ℃ and-80 ℃ as compared with example 1, and the difference gradually increased with the increase in storage temperature. When the sample is stored at a high temperature, the ribonuclease activity in the sample is high, and the beta-mercaptoethanol, the thiourea, the ethylene diamine tetraacetic acid disodium and the guanidine hydrochloride added in the embodiment of the invention can effectively inhibit the activity of the ribonuclease, slow down the degradation speed of the ribonuclease on RNA in the sample, help to maintain the biological activity of the RNA in the sample and prolong the storage time of the sample.

In conclusion, the RNA protective agent is a nontoxic sample storage solution which can be directly used, and the principle of the RNA protective agent is to inhibit the activity of the ribonuclease and protect RNA in a fresh sample from degradation. The RNA protective agent can effectively ensure that the RNA sample can be stored for a long time at 37 ℃ for 3 days, 18-25 ℃ for 7 days, 2-8 ℃ for 4 weeks and-20 ℃ or-80 ℃. Although the sample preserved by the method of repeated freeze thawing still enables the RNA to have high biological activity, the RNA contained in the sample preserved by the RNA protectant obtained by the invention can be used for all subsequent experiments about the RNA, and the damage to the sample caused by the repeated freeze thawing of the liquid preserved RNA sample is avoided. The RNA protective agent obtained by the invention does not influence the integrity and activity of RNA, and has important application value for the storage, the repeated use and the long-distance transportation of RNA.

The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.