Urine sample RNA stabilizing solution and preparation method thereof
阅读说明：本技术 尿液样本rna稳定液及制备方法 (Urine sample RNA stabilizing solution and preparation method thereof ) 是由 朱友杰 孔繁平 于 2019-11-12 设计创作，主要内容包括：本发明本发明公开了一种尿液样本RNA稳定液及制备方法,包括：0.01M-2M的二硫苏糖醇、2M-6M的异硫氰酸胍、1-200mM的乙二胺四乙酸、0.05-0.5mM的焦碳酸二乙酯和超纯水。本发明的制备步骤包括：在超纯水中加入一定量的上述化学试剂,用盐酸调节PH至4-4.2后定容,密封保存。本发明提供的尿液样本RNA稳定液,可以有效保持尿液样品中RNA的稳定性,可实现大体积尿液的保存和运输,并且适合后续RNA提取和荧光RT-PCR。(The invention discloses a urine sample RNA stabilizing solution and a preparation method thereof, wherein the urine sample RNA stabilizing solution comprises the following steps: 0.01M-2M dithiothreitol, 2M-6M guanidinium isothiocyanate, 1-200mM ethylenediaminetetraacetic acid, 0.05-0.5mM diethyl pyrocarbonate, and ultrapure water. The preparation method comprises the following steps: adding a certain amount of the chemical reagent into ultrapure water, adjusting the pH to 4-4.2 with hydrochloric acid, metering the volume, sealing and storing. The urine sample RNA stabilizing solution provided by the invention can effectively maintain the stability of RNA in a urine sample, can realize the storage and transportation of large-volume urine, and is suitable for subsequent RNA extraction and fluorescence RT-PCR.)
1. Urine sample RNA stabilizing solution, its characterized in that includes:
0.01M-2M dithiothreitol, 2M-6M guanidinium isothiocyanate, 1-200mM ethylenediaminetetraacetic acid and ultrapure water.
2. The urine sample RNA stabilizer according to claim 1, wherein:
the pH value of the urine sample RNA stabilizing solution is 3-5.
3. The urine sample RNA stabilizer according to claim 1, wherein:
the urine sample RNA stabilizing solution consists of 0.1M dithiothreitol, 2M guanidinium isothiocyanate, 0.2mM diethyl pyrocarbonate, 100mM ethylene diamine tetraacetic acid and ultrapure water.
4. The method for preparing the RNA stabilizing solution for the urine sample according to claim 1, which comprises the following steps:
the method comprises the following steps: preparing AmL ultrapure water;
step two: weighing dithiothreitol, guanidinium isothiocyanate and ethylenediamine tetraacetic acid, adding into the ultrapure water obtained in the first step, and stirring for dissolving;
step three: weighing diethyl pyrocarbonate, adding the diethyl pyrocarbonate into the solution obtained in the second step, and uniformly mixing;
step four: adjusting pH to 4-4.2 with hydrochloric acid;
step four: fixing the volume to 2AmL, and sealing and storing;
wherein, the stable liquid is ensured to meet the following conditions: 0.01M-2M dithiothreitol, 2M-6M guanidinium isothiocyanate, 1-200mM ethylenediaminetetraacetic acid, 0.05-0.5mM diethylpyrocarbonate.
Technical Field
The invention relates to RNA sample stabilizing solution, in particular to urine sample RNA stabilizing solution and a preparation method thereof.
Background
Liquid biopsy, which is a technique for detecting and acquiring tumor-related information by using body fluids (blood, urine, and other body fluids) of a human body as a specimen source, is a simple and non-invasive tissue biopsy substitute method. Has the advantages of good patient compliance, good specificity, low heterogeneity, repeated material taking and the like. Therefore, the early screening of cancer by liquid biopsy technology has very good development prospect.
Urine is produced by the kidneys, stored via the ureters into the bladder, and then excreted via the urethra. Due to the physiological characteristics of urine, urine samples are an excellent source of liquid biopsy samples for urinary system tumors, and therefore urinary system tumors such as bladder cancer, prostate cancer and the like are all dedicated to search for appropriate molecular markers in urine samples. Particularly, prostate cancer is difficult to be detected early by other means because the prostate gland is in the body and has a small volume, and when the prostate gland is cancerated, the prostate gland is directly communicated with the urethra, and prostate components can be detected from urine, so that nucleic acid molecules in the urine can be used as biomarkers of the prostate cancer.
However, urine has a large volume and a low nucleic acid concentration, wherein prostate-derived components are fewer, particularly prostate-specific RNA molecules, the content of the prostate-specific RNA molecules is not high, and the RNA is easy to degrade and cannot be detected.
Disclosure of Invention
The invention aims to solve the problem that the existing urine sample RNA stabilizing solution cannot store urine RNA for a long time, provides the urine sample RNA stabilizing solution, can effectively keep the stability of the RNA in a urine sample, can realize the storage and transportation of large-volume urine, and is suitable for subsequent RNA extraction and fluorescence RT-PCR.
The technical scheme for realizing the aim of the invention is that the urine sample RNA stabilizing solution comprises: 0.01M-2M dithiothreitol, 2M-6M guanidinium isothiocyanate, 1-200mM ethylenediaminetetraacetic acid and ultrapure water.
The urine sample RNA stabilizing solution also comprises 0.05-0.5mM diethyl pyrocarbonate.
The pH value of the urine sample RNA stabilizing solution is 3-5.
The urine sample RNA stabilizing solution is preferably composed of 0.1M dithiothreitol, 2M guanidine isothiocyanate, 0.2mM diethyl pyrocarbonate, 100mM ethylene diamine tetraacetic acid and ultrapure water.
The preparation method of the urine sample RNA stabilizing solution comprises the following steps:
the method comprises the following steps: preparing AmL ultrapure water;
step two: weighing dithiothreitol, guanidinium isothiocyanate and ethylenediamine tetraacetic acid, adding into the ultrapure water obtained in the first step, and stirring for dissolving;
step three: weighing diethyl pyrocarbonate, adding the diethyl pyrocarbonate into the solution obtained in the second step, and uniformly mixing;
step four: adjusting pH to 4-4.2 with hydrochloric acid;
step four: fixing the volume to 2AmL, and sealing and storing;
wherein, the stable liquid is ensured to meet the following conditions: 0.01M-2M dithiothreitol, 2M-6M guanidinium isothiocyanate, 1-200mM ethylenediaminetetraacetic acid, 0.05-0.5mM diethylpyrocarbonate.
By adopting the technical scheme, the invention has the following technical effects:
1. the urine sample RNA stabilizing solution provided by the invention can effectively keep the stability of RNA in a urine sample, can realize the storage and transportation of large-volume urine, and is suitable for subsequent RNA extraction and fluorescence RT-PCR.
2. The invention adopts dithiothreitol as a reducing agent, which can reduce protein disulfide bonds, thereby changing the conformation of RNA enzyme in urine, inactivating the RNA enzyme and ensuring the long-term preservation of RNA.
3. The method adopts diethyl pyrocarbonate as an RNA enzyme inactivator, and can effectively and specifically combine with an imidazole ring of an active group histidine of RNA enzyme, thereby denaturing and inactivating the RNA enzyme and ensuring the preservation of RNA.
4. The invention adopts ethylenediamine tetraacetic acid as a divalent cation chelating agent, and can competitively combine dicationins necessary for RNA enzyme, so that the activity of RNA enzyme is reduced, and the decomposition of RNA is slowed down.
5. The invention adopts guanidinium isothiocyanate as a strong denaturing agent, and the guanidinium isothiocyanate with high concentration can destroy cell membranes, so that RNA is separated from protein, and the efficiency and detection of subsequent RNA extraction are greatly improved.
6. The invention adopts ultrapure water to provide a liquid environment for the stabilizer, and is convenient for later-stage production and application.
Drawings
In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the present disclosure taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a PSA mRNA amplification curve after fluorescent RT-PCR of urine added with urine RNA stabilizer;
FIG. 2 is a graph showing the amplification curve of PSA mRNA after fluorescent RT-PCR in urine without the addition of urine RNA stabilizer.
Detailed Description
(example 1)
The foregoing description is only an overview of the technical solutions of the present invention, and the embodiments of the present invention are described below in order to make the technical means of the present invention more clearly understood and to make the above and other objects, features, and advantages of the present invention more clearly understandable.
The invention provides a urine sample RNA stabilizing solution, which is used for solving the problems that the existing urine sample RNA stabilizing solution cannot store urine RNA for a long time and cannot store and transport large-volume urine, and in order to solve the problems, the general idea of the invention is as follows:
the method comprises the following steps: preparing AmL ultrapure water;
step two: weighing dithiothreitol, guanidinium isothiocyanate and ethylenediamine tetraacetic acid, adding into the ultrapure water obtained in the first step, and stirring for dissolving;
step three: weighing diethyl pyrocarbonate, adding the diethyl pyrocarbonate into the solution obtained in the second step, and uniformly mixing;
step four: adjusting pH to 4-4.2 with hydrochloric acid;
step four: fixing the volume to 2AmL, and sealing and storing;
wherein, the stable liquid is ensured to meet the following conditions: 0.01M-2M dithiothreitol, 2M-6M guanidinium isothiocyanate, 1-200mM ethylenediaminetetraacetic acid, 0.05-0.5mM diethylpyrocarbonate.
The technical solutions of the present invention are described in detail below with reference to the drawings and specific embodiments, and it should be understood that the specific features in the embodiments and examples of the present invention are described in detail in the technical solutions of the present application, and are not limited to the technical solutions of the present application, and the technical features in the embodiments and examples of the present application may be combined with each other without conflict.
(example 1)
Preparing a urine sample RNA stabilizing solution, and controlling the contents of the components as follows: dithiothreitol 0.1M, guanidinium isothiocyanate 2M, diethyl pyrocarbonate 0.2mM, ethylenediaminetetraacetic acid 100 mM; the stabilizing solution is prepared according to the steps. In the same urine sample, 90mL of urine is respectively taken, 10mL of the stabilizer solution is added into the experimental group, 10mL of the ultrapure water is added into the control group, the two groups are evenly mixed and divided into 5 parts, after the two groups are respectively stored at room temperature for 0, 1, 3, 5 and 7 days, the total RNA of the urine is extracted by using a TRIZOL method, and the content of the RNA is measured by using a NanoDrop (spectrophotometer), and the results are shown in Table 1:
TABLE 1
As can be seen from Table 1, the control group and the experimental group were stored at room temperature for 0 day, 1 day, 3 days, 5 days, and 7 days, and total RNA was extracted with the same volume of urine by the same method to determine the RNA content, wherein the RNA extraction amount of the control group was significantly decreased after one day of storage at room temperature, almost no RNA was detected after three days, and the RNA extraction amount of the experimental group was not significantly different after 7 days of storage at room temperature.
(example 2)
Preparing a urine sample RNA stabilizing solution, and controlling the contents of the components as follows: dithiothreitol 0.01M, guanidinium isothiocyanate 2M, diethyl pyrocarbonate 0.05mM, ethylenediaminetetraacetic acid 200 mM; the stabilizing solution is prepared according to the steps. In the same urine sample, 90mL of urine is respectively taken, 10mL of the stable solution is added into the experimental group, 10mL of ultrapure water is added into the control group, the two groups are evenly mixed and divided into 5 parts, after the two groups are respectively stored at room temperature for 0, 1, 3, 5 and 7 days, the total RNA of the urine is extracted by using a TRIZOL method, the content of the RNA is measured by using NanoDrop, and the results are shown in Table 2:
TABLE 2
(example 3)
Preparing a urine sample RNA stabilizing solution, and controlling the contents of the components as follows: dithiothreitol 2M, guanidinium isothiocyanate 6M, diethyl pyrocarbonate 0.5mM, and ethylenediaminetetraacetic acid 100 mM. The stabilizing solution is prepared according to the steps. In the same urine sample, 90mL of urine is respectively taken, 10mL of the stable solution is added into the experimental group, 10mL of ultrapure water is added into the control group, the two groups are evenly mixed and divided into 5 parts, after the two groups are respectively stored at room temperature for 0, 1, 3, 5 and 7 days, the total RNA of the urine is extracted by using a TRIZOL method, the content of the RNA is measured by using NanoDrop, and the results are shown in Table 3:
TABLE 3
(example 4)
Preparing a urine sample RNA stabilizing solution, and controlling the contents of the components as follows: dithiothreitol 2M, guanidinium isothiocyanate 6M, diethyl pyrocarbonate 0.05mM, and
TABLE 4
(example 5)
Preparing a urine sample RNA stabilizing solution, and controlling the contents of the components as follows: dithiothreitol 1M, guanidinium isothiocyanate 4M, diethyl pyrocarbonate 0.3mM, and ethylenediaminetetraacetic acid 100 mM. The stabilizing solution is prepared according to the steps. In the same urine sample, 90mL of urine is respectively taken, 10mL of the stabilizer solution is added into the experimental group, 10mL of the ultrapure water is added into the control group, the two groups are evenly mixed and divided into 5 parts, the two parts are respectively stored at room temperature for 0, 1, 3, 5 and 7 days, the total RNA of the urine is extracted by using a TRIZOL method, the content of the RNA is measured by using NanoDrop, and the results are shown in Table 5:
TABLE 5
Preparing a urine sample RNA stabilizing solution, and controlling the contents of the components as follows: dithiothreitol 0.1M, guanidinium isothiocyanate 2M, diethyl pyrocarbonate 0.2mM, ethylenediaminetetraacetic acid 100 mM; the stabilizing solution is prepared according to the steps. In the same urine sample, respectively taking 90mL of urine, adding 10mL of LRNA stabilizing solution into an experimental group, adding 10mL of ultrapure water into a control group, uniformly mixing the two groups, dividing the two groups into 5 parts, respectively storing the two parts at room temperature for 0, 1, 3, 5 and 7 days, extracting nucleic acid, and detecting the mRNA of the Prostate Specific Antigen (PSA) gene by one-step fluorescence RT-PCR.
FIG. 1 shows the amplification curve of the control group, in which the Ct value is significantly decreased after 1 day of storage and is not detected after 3 days; for example, fig. 2 is an amplification curve of the experimental group, the amplification curve of the experimental group is an obvious exponential amplification S-type curve, and Ct values are very similar, which indicates that PSA RNA in the sample is always stable, and thus it can be seen that the stability of prostate-derived RNA in the urine sample can be protected for at least 7 days.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.