Model substance and quality assurance method

Abstract

A model substance according to the present disclosure comprises a mixture prepared by mixing biological samples collected from eight or more subjects of the same biological species and can be used for quantitative analysis of small RNA.

Description

Standard materials and quality assurance methods

[0001] This disclosure relates to reference materials and quality assurance methods.

[0002] Small RNAs (SMOs) are low-molecular-weight RNAs that exist in living organisms, and their structure and function are being increasingly studied. The expression patterns of SMOs are known to be associated with various diseases, including cancer and cardiovascular disease. Therefore, analyzing SMOs is extremely useful for disease diagnosis, treatment, and understanding disease progression.

[0003] Currently used small RNA analysis methods include qRT-PCR, microarrays, and next-generation sequencing (NGS). In any of these technologies, it is desirable to consistently obtain consistent data across different time periods, instruments, or experimental environments, and standard materials are required for quality assurance, analytical calibration, and anomaly detection.

[0004] Here, a standard substance is a substance whose purity, composition, properties, etc., are precisely defined and used as a reference in experiments and measurements. It is widely used in experiments and industry for quality assurance, analytical calibration, and anomaly detection.

[0005] Patent Document 1 discloses a method in which nucleic acids with a nucleic acid length of 200 bases or more are used as standard substances, a fixed amount of this standard substance is added to a fixed amount of each sample, nucleic acids are extracted from the samples, the amount of each target small RNA and standard substance extracted is measured, and the expression level of the target small RNA is corrected using the measured amount of the extracted standard substance.

[0006] Patent Document 1: International Publication No. 2016 / 084848

[0007] A mixture of chemically synthesized RNAs is used as a standard material for RNA. However, there are no commercially available products that are used as a standard material for small RNA.

[0008] While it might be possible to use a mixture of chemically synthesized small RNAs as a standard substance for small RNAs, similar to a mixture of chemically synthesized RNAs, this would require constructing a new chemical synthesis system each time the target of measurement changes. Furthermore, it is difficult to chemically synthesize dozens to hundreds of types of small RNAs with consistent quality and to mix them at a constant concentration each time.

[0009] In other words, as a standard material for small RNA, it is required to have stability that allows for long-term use at the same quality, comprehensiveness that includes multiple types of small RNA, and reproducibility that ensures consistent quality across multiple manufacturing lots.

[0010] This disclosure relates to a standard material and quality assurance method for the quantitative analysis of small RNAs that offers excellent stability, comprehensiveness, and reproducibility.

[0011] Means for solving the above problems include the following embodiments: <1> A standard substance for quantitative analysis of small RNA, comprising a mixture prepared by mixing biological samples collected from eight or more individuals of the same species. <2> The standard substance according to <1>, wherein the mixture is prepared by mixing biological samples collected from 16 or more individuals of the same species. <3> The standard substance according to <1>, wherein the mixture is prepared by mixing biological samples collected from 32 or more individuals of the same species. <4> The standard substance according to any one of <1> to <3>, wherein the species is human. <5> The standard substance according to any one of <1> to <4>, wherein the mixture is serum or plasma. <6> The standard substance according to <5>, prepared by adding a substance having a nucleic acid preservation effect to the mixture. <7> The standard substance according to any one of <1> to <6>, wherein RNA containing small RNA is purified from the mixture and the RNA containing the small RNA is extracted. <8> A standard substance according to any one of <1> to <7>, wherein the same biological species is the same in at least one of sex and age distribution. <9> A quality assurance method for quantitatively analyzing the expression level of small RNA using a standard substance consisting of a mixture prepared by mixing biological samples obtained from eight or more individuals of the same biological species. <10> The quality assurance method according to <9>, wherein the biological species is human. <11> The quality assurance method according to <10>, wherein if the male-to-female ratio of eight or more human individuals exceeds 70%, hsa-miR-122 is excluded and the expression level of small RNA is quantitatively analyzed.

[0012] This disclosure makes it possible to provide standard materials and quality assurance methods for the quantitative analysis of small RNAs that offer excellent stability, comprehensiveness, and reproducibility.

[0013] This figure shows the number of samples mixed and the number of samples prepared in Example 1. This figure shows the correlation between individual samples in Example 1. This figure shows the correlation between each sample when 16 types of samples are prepared by pooling samples from 4 people in Example 1. This figure shows the correlation between each sample when 10 types of samples are prepared by pooling samples from 8 people in Example 1. This figure shows the correlation between each sample when 10 types of samples are prepared by pooling samples from 16 people in Example 1. This figure shows the correlation between each sample when 10 types of samples are prepared by pooling samples from 32 people in Example 1. This figure shows the correlation between the average values ​​of miRNA expression levels by gender in Example 2. This figure shows the difference in miRNA expression levels by gender in Example 2. This figure shows the correlation when hsa-miR-122 is excluded in Example 2. This figure shows the correlation between the average values ​​of miRNA expression levels by age group in Example 3.

[0014] Next, embodiments of the present disclosure will be described in detail with reference to the drawings.

[0015] An example of an embodiment relating to the technology of this disclosure will be described below with reference to the drawings. Components and processes that perform the same operation, action, or function will be given the same reference numerals throughout the drawings, and redundant explanations may be omitted as appropriate. Each drawing is only a schematic representation to the extent that the technology of this disclosure can be fully understood. Therefore, the technology of this disclosure is not limited to the illustrated examples. Furthermore, in this embodiment, explanations of configurations not directly related to this disclosure or well-known configurations may be omitted.

[0016] The inventors of this disclosure have found that, in order to quantitatively analyze the expression level of small RNA, a standard substance containing all small RNAs present in a given species can be prepared by mixing biological samples collected from eight or more individuals of the same species. They have also found that by mixing biological samples collected from eight or more individuals of the same species, a standard substance of the same quality can be prepared even when using biological samples collected from eight or more different individuals of the same species. Furthermore, they have found that by mixing biological samples collected from eight or more individuals of the same species, a standard substance of the same quality can be prepared even when using new biological samples collected from eight or more different individuals of the same species several years or decades later. In other words, they have found that a standard substance for the quantitative analysis of small RNA with excellent stability, comprehensiveness, and reproducibility can be prepared.

[0017] [Standard Material] The standard material in this disclosure is a mixture prepared by mixing biological samples collected from eight or more individuals of the same species, used to quantitatively analyze the expression levels of small RNAs of multiple identical species.

[0018] Furthermore, the standard substance of this disclosure is preferably a mixture prepared by mixing biological samples collected from 16 or more individuals of the same biological species, and more preferably a mixture prepared by mixing biological samples collected from 32 or more individuals of the same biological species.

[0019] Examples of biological species include animals and plants. Examples of animals include humans and non-human animals. Examples of non-human animals include mammals other than humans (e.g., monkeys, dogs, cats, mice, rats, rabbits, cows, horses, pigs, and sheep).

[0020] Examples of biological samples include serum, plasma, whole blood (also called blood), urine, tears, saliva, sweat, semen, bone marrow fluid, lymph, tissue fluid, body cavity fluids (e.g., pleural fluid, ascites), cerebrospinal fluid, amniotic fluid, vaginal fluid, nasal mucus, feces, and tissue.

[0021] Furthermore, if the species is human, it is preferable to use a human biological sample that has the same sex and age distribution.

[0022] Furthermore, the standard material of this disclosure may be prepared by adding a preservative, which is a substance that preserves nucleic acids, to the mixture described above. This makes it possible to extend the shelf life of the standard material.

[0023] The preservative should be added to the separated serum or plasma within four hours of blood collection. From the viewpoint of measuring small RNA expression levels with higher accuracy, the preservative should preferably be added within two hours, and more preferably within one hour.

[0024] Preservatives are typically liquids and contain preservative components for the stable storage of small RNA. In addition to preservative components, preservatives may also contain other additives. Examples of preservative components include chaotropic agents, reducing agents, buffers, surfactants, and chelating agents.

[0025] Examples of chaotropic agents include guanidinium salts, urea, and iodide salts. Examples of guanidinium salts include guanidine thiocyanate, guanidine thioisocyanate, guanidine hydrochloride, guanidine hydrochloride, guanidinium chloride, guanidinium iodide, sodium iodide, and potassium iodide.

[0026] Examples of reducing agents include thioglycerin, mercaptoethanol, dithiothreitol, tri(2-carboxyethyl)phosphine, and dithiothreitol (DTT).

[0027] Examples of buffering agents include sodium acetate, sodium citrate, sodium bicarbonate, tris(hydroxymethyl)aminomethane, sodium hydrogen phosphate, sodium dihydrogen phosphate, and so-called Good's buffering agents.

[0028] Examples of surfactants include sodium lauroyl sarcosinate, sodium dodecyl sulfate, polysorbate (Tween (registered trademark)), lauryldimethylamine oxide, cetyltrimethylammonium bromide, polyethoxylated alcohol, polyoxyethylene, sorbitan, octoxynol (Triton X-100 (registered trademark)), N,N-dimethyldodecylamine-N-oxide, hexadecyltrimethylammonium bromide, polyoxyl 10 lauryl ether, sodium deoxycholate, sodium cholate, polyoxyl castor oil (Cremophor (registered trademark)), nonylphenol ethoxylate (Tergitol (registered trademark)), cyclodextrin, lecithin, methylbenzethonium chloride (Hyamine (registered trademark)), etc.

[0029] Examples of chelating agents include EDTA, citric acid, phytic acid, gluconic acid, etc.

[0030] Other additives include methylacetamide.

[0031] Each component included in the preservative may be used alone or in combination of two or more.

[0032] Among them, the preservative preferably contains a guanidium salt. Since the guanidium salt has the effect of inactivating RNase present in biological samples such as serum, it can effectively stabilize RNA. In addition, the guanidium salt is also useful in that it can detoxify the infectivity of serum and the like.

[0033] In one aspect, the preservative may contain a chaotropic agent, a reducing agent, a buffer, and N-methylacetamide, and may not contain other components. In a further aspect, the preservative may contain a chaotropic agent, a reducing agent, and a buffer, and may not contain other components. In one aspect, the preservative may not contain a chelating agent (for example, EDTA).

[0034] Also, the reference substance of the present disclosure may be prepared by purifying an RNA solution containing small RNA from the above-described mixture and extracting RNA containing small RNA.

[0035] Other examples of small RNAs include microRNA (hereinafter referred to as miRNA), piRNA, and tsRNA.

[0036] [Quality Assurance Method] A quality assurance method in one embodiment of the present disclosure includes quantitatively analyzing the expression level of small RNA using a standard substance consisting of a mixture prepared by mixing biological samples obtained from eight or more individuals of the same species. According to the quality assurance method of this embodiment, it is possible to quantitatively analyze the expression level of small RNA using the standard substance.

[0037] Specifically, using the aforementioned standard materials, it is possible to, for example, guarantee the quality of instruments that quantitatively analyze the expression level of small RNAs such as NGS, correct the expression level data of small RNAs contained in biological samples being analyzed, and detect abnormalities in small RNAs contained in biological samples being analyzed.

[0038] Furthermore, the quality assurance method disclosed herein may include excluding hsa-miR-122 and quantitatively analyzing the expression level of small RNAs when the male-to-female ratio or the female-to-male ratio of eight or more human individuals exceeds 70%. This makes it possible to quantitatively analyze the expression level of small RNAs without considering differences based on sex.

[0039] As a quality assurance method for quantitatively analyzing the expression levels of small RNAs, various methods, including known techniques, can be employed, provided that the expression levels of multiple small RNAs are measured using the aforementioned standard substances by methods such as NGS, quantitative PCR, and flow cytometry, and the results (i.e., small RNA data) can be obtained. The small RNA data may be absolute quantitative values ​​quantified as absolute values, or relative expression levels quantified as relative expression levels.

[0040] The present disclosure will be further described below with reference to examples, but the present disclosure is not limited to the following examples unless it exceeds the spirit of the disclosure.

[0041] In the following description, a case of performing quantitative analysis of miRNA by measuring the expression level of miRNA from the serum of a human subject will be used for explanation.

[0042] [Example 1] <Analysis of the number of individuals used as a standard substance> Blood was collected from 64 subjects, and the collected blood was centrifuged to obtain serum. Then, as shown in FIG. 1, 64 types of individual samples, 16 types of samples obtained by mixing sera for 4 persons, 10 types of samples obtained by mixing sera for 8 persons, 10 types of samples obtained by mixing sera for 16 persons, 10 types of samples obtained by mixing sera for 32 persons, and 1 type of sample obtained by mixing sera for 64 persons were prepared. Each sample was prepared by mixing equal amounts of sera from each person. Note that the samples obtained by mixing sera for 4 persons were all composed of sera from different individuals, and there were individuals whose sera were used repeatedly in the samples obtained by mixing sera for 8 persons, 16 persons, or 32 persons. Then, RNA was extracted from the individual samples and the prepared samples, and the relative expression level of miRNA was measured by NGS to evaluate whether reproducibility as a standard substance can be obtained by pooling sera for a certain number of persons.

[0043] As an evaluation index for reproducibility, the coefficient of determination R of the quantitative values (log ｉ , ２ RPM (Read Per Million)) of the top 1 to 50 miRNAs in descending order of the expression level of miRNA was used. The threshold for reproducibility was set as the coefficient of determination R ２ = 0.95. This is because the coefficient of determination R ２ which is the measurement accuracy including random errors and differences between runs when calculating the expression level of miRNA by NGS, is about 0.94. Note that the closer the coefficient of determination R ２ is to 1, the higher the correlation between the corresponding samples and the higher the reproducibility. On the other hand, the farther the coefficient of determination R ２ is from 1, the lower the correlation between the corresponding samples and the lower the reproducibility. ２ The coefficient of determination R

[0044] was calculated based on the following formula. In the following formula, the measured value y ２ is the expression level of miRNA in one sample, and the predicted value ax i is used to fit the expression level of miRNA in one sample, and the predicted value ax ｉ+b represents the miRNA expression level of the other sample.

[0045] Also, the coefficient of determination ２ This is sometimes called the rate of contribution, and can also be shown as follows.

[0046] Also, the coefficient of determination ２ The square root of this is sometimes called the correlation coefficient R.

[0047] Figure 2 shows the correlation of miRNA expression levels in individual samples from 64 subjects. In Figure 2, the coefficient of determination R between individual samples is shown. ２ The coefficient of determination R is 0.95 or higher, and ２ The larger the value of R, the darker the color of the cell, and the coefficient of determination R ２ Cells with a value less than 0.95 are shown in white. The coefficient of determination R indicates the correlation between individual samples. ２ The coefficient of determination R is 0.74 to 0.99. ２ The average was 0.93. Individual differences were observed between individual samples, and the coefficient of determination R ２ It was confirmed that there was variability.

[0048] Figure 3 shows the correlation of miRNA expression levels in samples 4-1 to 4-16, which are pooled serum samples from four individuals. The coefficient of determination R between samples 4-1 to 4-16, which are mixed serum samples from four individuals, is shown. ２ The values ​​ranged from 0.93 to 0.99. That is, the inter-sample coefficient of determination R ２ It was confirmed that some values ​​were less than 0.95.

[0049] Figure 4 shows the correlation of miRNA expression levels in samples 8-1 to 8-10, which are pooled serum samples from eight individuals. The coefficient of determination R between samples 8-1 to 8-10, which are mixed serum samples from eight individuals, is shown. ２ The values ​​ranged from 0.96 to 0.99. That is, the coefficient of determination R between all samples pooled from serum samples of 8 individuals. ２ It was confirmed that the value exceeded 0.95.

[0050] Figure 5 shows the correlation of miRNA expression levels in samples 16-1 to 16-10, which were pooled from serum samples of 16 individuals. The coefficient of determination R for samples 16-1 to 16-10, which were mixed from serum samples of 16 individuals. ２ The coefficient of determination R was 0.98 to 0.99. That is, the coefficient of determination R between all samples pooled from serum samples of 16 individuals. ２ It was confirmed that the value exceeded 0.95.

[0051] Figure 6 shows the correlation of miRNA expression levels in samples 32-1 to 32-10, which were pooled from serum samples of 32 individuals. The coefficient of determination R for samples 32-1 to 32-10, which were mixed from serum samples of 32 individuals. ２ The values ​​ranged from 0.99 to 1.00. That is, the coefficient of determination R between all samples pooled from serum samples of 32 individuals. ２ It was confirmed that the value exceeded 0.95.

[0052] <Results> By pooling serum samples from 8 or more people to create a standard substance, the coefficient of determination R ２ It was confirmed that the reproducibility threshold of 0.95 or higher could be set, enabling the creation of a standard substance with excellent reproducibility. Furthermore, it was confirmed that the greater the number of serum samples pooled, the better the reproducibility. In other words, it was confirmed that pooling serum from at least eight individuals provides the reproducibility necessary to use it as a standard substance.

[0053] [Example 2] <Analysis of differences by sex> RNA was extracted from serum collected from 13 women and 51 men out of 64 test subjects, and the expression levels of miRNAs were measured. Figure 7 shows the correlation between the average expression levels of each miRNA in the 13 women and the average expression levels of each miRNA in the 51 men. Figure 8 shows the difference between the average expression levels of each miRNA in the 13 women and the average expression levels of each miRNA in the 51 men, with the difference in miRNA expression levels between men and women displayed on a logarithmic scale. In other words, in this example, by calculating the average values ​​for each miRNA in the 13 women and the 51 men, the correlation between the expression levels of miRNAs in a pooled sample of serum from the 13 women and a pooled sample of serum from the 51 men is simulated. The same applies to Example 3, which will be described later.

[0054] The coefficient of determination R of the mean miRNA expression levels of 13 women and the mean miRNA expression levels of 51 men. ２ The value was 0.98. Furthermore, as shown in Figures 7 and 8, it was confirmed that there was a significant difference in the expression level of hsa-miR-122 between sexes compared to the expression levels of other miRNAs.

[0055] Figure 9 shows the correlation between the expression levels of miRNAs from 13 women and 51 men, when the hsa-miR-122 miRNA was excluded from the analysis. By excluding hsa-miR-122 from the analysis, the coefficient of determination R ２ We were able to reduce the value from 0.98 to 0.99. In other words, the influence of gender differences was reduced.

[0056] <Results> Even including hsa-miR-122, which shows a large gender difference, the coefficient of determination R ２ It was confirmed that the reproducibility threshold of 0.95 or higher could be achieved, and that there are no practical problems even if standard materials are prepared without considering the male-female ratio (sex). However, it was confirmed that if the male-female ratio is significantly skewed, it is more preferable to perform the analysis using data excluding hsa-miR-122.

[0057] [Example 3] <Analysis of differences by age> RNA was extracted from serum collected from 16 people in their 20s, 16 people in their 30s, 16 people in their 40s, and 13 people in their 50s out of 61 test subjects, and the expression levels of miRNAs were measured. Figure 10 shows the correlation between the average expression levels of each miRNA in the 16 people in their 20s, the average expression levels of each miRNA in the 16 people in their 30s, the average expression levels of each miRNA in the 16 people in their 40s, and the average expression levels of each miRNA in the 13 people in their 50s.

[0058] <Results> As shown in Figure 10, the coefficient of determination R of the mean miRNA expression levels between different age groups is ２ The values ​​ranged from 0.99 to 1.00. Therefore, it was confirmed that there are no practical problems in preparing standard materials without considering age. However, it was confirmed that it is more preferable to perform the analysis using data excluding hsa-miR-122.

[0059] As mentioned above, similar to gender and age, certain miRNAs may differ depending on race, living standards, and living environment. Therefore, it is preferable to use biological samples collected from populations with similar races, living standards, and living environments as standard materials. Furthermore, if a specific miRNA is identified as the cause, the influence of differences due to race, living standards, and living environment can be reduced by excluding that specific miRNA from the analysis. Therefore, it is preferable to collect information on race, living standards, and living environment when collecting biological samples to be used as standard materials.

[0060] From the above, it was confirmed that a standard substance and quality assurance method for the quantitative analysis of small RNAs with excellent stability, comprehensiveness, and reproducibility can be obtained.

[0061] In Example 1 described above, the reproducibility for use as a standard substance was evaluated using samples mixed from the serum of 4 people, 8 people, 16 people, and 32 people. However, as in Examples 2 and 3 described above, the correlation may also be evaluated by calculating the average value of the miRNA expression levels of 4, 8, 16, and 32 people, respectively. Similarly, in Examples 2 and 3 described above, the correlation may also be evaluated using samples mixed from serum by sex or age group, as in Example 1 described above.

[0062] The disclosure of Japanese Patent Application No. 2024-188787, filed on 28 October 2024, is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually noted to be incorporated by reference.

[0063] [Note] Preferred embodiments of this disclosure are described below. (Embodiment 1) A standard substance for quantitative analysis of small RNA, comprising a mixture prepared by mixing biological samples collected from eight or more individuals of the same species.

[0064] (Aspect 2) The standard substance according to Aspect 1, wherein the mixture is prepared by mixing biological samples collected from 16 or more individuals of the same biological species.

[0065] (Aspect 3) The mixture is a standard substance according to Aspect 1, prepared by mixing biological samples collected from 32 or more individuals of the same biological species.

[0066] (Aspect 4) The standard substance according to any one of aspects 1 to 3, wherein the species of organism is human.

[0067] (Aspect 5) The standard substance according to any one of aspects 1 to 4, wherein the mixture is serum or plasma.

[0068] (Aspect 6) A standard substance according to aspect 5, prepared by adding a substance having nucleic acid preservation effect to the mixture.

[0069] (Aspect 7) A standard substance according to any one of aspects 1 to 6, obtained by purifying RNA containing small RNA from the mixture and extracting the RNA containing the small RNA.

[0070] (Aspect 8) The standard substance according to any one of aspects 1 to 7, wherein the same biological species has the same sex and age distribution.

[0071] (Aspect 9) A quality assurance method for quantitatively analyzing the expression level of small RNA using a standard substance consisting of a mixture prepared by mixing biological samples obtained from eight or more individuals of the same species.

[0072] (Aspect 10) The quality assurance method according to aspect 9, wherein the biological species is human.

[0073] (Aspect 11) The quality assurance method according to aspect 10, wherein when the ratio of males or females among eight or more human individuals exceeds 70%, hsa-miR-122 is excluded and the expression level of small RNA is quantitatively analyzed.

Claims

1. A standard substance for the quantitative analysis of small RNA, consisting of a mixture prepared by mixing biological samples collected from eight or more individuals of the same species.

2. The standard substance according to claim 1, wherein the mixture is prepared by mixing biological samples collected from 16 or more individuals of the same biological species.

3. The standard substance according to claim 1, wherein the mixture is prepared by mixing biological samples collected from 32 or more individuals of the same biological species.

4. The standard substance according to claim 1, wherein the biological species is human.

5. The standard substance according to claim 1, wherein the mixture is serum or plasma.

6. The standard substance according to claim 5, which is prepared by adding a substance having nucleic acid preservation effect to the mixture.

7. The standard substance according to claim 1, wherein RNA containing small RNA is purified from the mixture, and the RNA containing small RNA is extracted.

8. The standard substance according to claim 1, wherein the same biological species has the same sex and age distribution.

9. A quality assurance method for quantitatively analyzing the expression level of small RNA using a standard substance consisting of a mixture prepared by mixing biological samples obtained from eight or more individuals of the same species.

10. The quality assurance method according to claim 9, wherein the biological species is human.

11. The quality assurance method according to claim 10, wherein when the male-to-female ratio of eight or more human individuals exceeds 70%, hsa-miR-122 is excluded and the expression level of small RNA is quantitatively analyzed.

Images