Method for detecting microrna in biological sample, composition of hydrochloride of neutral amino acid, and container

Abstract

To address the problems in which: after a biological sample containing microRNA is collected, if the collected sample undergoes a storage step, its detectable amount decreases; a crystalline neutral amino acid hydrochloride takes a relatively long time to dissolve in water; and the neutral amino acid hydrochloride is an acidic substance that causes skin irritation and mucosal irritation, thus requiring safety precautions when being handled.SOLUTION: The present invention is a method for detecting microRNA in a biological sample and is characterized by comprising a step for mixing acid in a biological sample, a step for storing the microRNA for a prescribed time, a step for recovering the microRNA from the biological sample, and a step for detecting the microRNA. The present invention also provides a homogenous semi-solid composition comprising a hydrochloride of a neutral amino acid, water, and an alcohol.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for detecting microRNA in a biological sample, and to a composition comprising a hydrochloride salt of a neutral amino acid. [Background technology]

[0002] With the recent development of genetic analysis technology, attempts have been made to detect microRNAs in biological samples and use them for disease diagnosis, treatment, etc. Detecting disease-related microRNAs in biological samples such as blood may enable earlier detection of the disease.

[0003] On the other hand, compared to general RNA, which has been the subject of various studies to date, microRNAs contained in biological samples such as blood are present in extremely small amounts compared to other RNAs and are easily degraded by RNases contained in biological samples. MicroRNAs, which are used as markers in cancer diagnosis, are present extracellularly in serum and body fluids and are therefore particularly susceptible to the effects of RNases compared to general intracellular RNAs.

[0004] For this reason, biological samples used for microRNA detection are usually used for detection immediately after acquisition. Alternatively, after acquisition, the biological sample must be stored in a state that suppresses microRNA degradation by methods such as freezing before use for detection. Thus, in the detection of microRNA, more care must be taken in handling biological samples than in the case of other RNAs.

[0005] In industrial settings, the clinical site where biological samples are obtained and the testing institution where microRNAs are detected are often far apart, and the biological samples must be stored for long periods of time, from several hours to several days, while they are transported from the clinical site to the testing institution.

[0006] Biological samples used for microRNA detection are generally stored frozen at ultra-low temperatures below -70°C. However, the freezing equipment and transportation materials required to keep biological samples at ultra-low temperatures are expensive, and there is a need for a method other than freezing to store biological samples used for microRNA detection for long periods of time.

[0007] In addition to freezing, methods to suppress RNA degradation in biological samples include adding guanidinium salts to the biological sample to denature RNases and adding synthetic small molecule inhibitors. However, a more effective and convenient method is to suppress RNA degradation by adjusting the pH of the biological sample to a pH range where RNase activity is reduced.

[0008] Patent Document 1 describes a method for utilizing RNA by adjusting the pH of a biological sample to 4.0 or below, at which point the activity of RNase is reduced, thereby suppressing RNA degradation, and then inactivating the RNase, and then adjusting the pH of the biological sample to greater than 6.0.

[0009] Neutral amino acid hydrochlorides, including glycine hydrochloride, are compounds primarily used in biochemistry. In particular, due to their ability to reversibly dissociate antibody-antigen bonds, they are used for antibody elution in affinity purification, washing of primary antibodies in multiplex immunostaining, and testing for autoantibodies bound to red blood cells.

[0010] It is also used in fields other than biochemistry, and because it has the property of solubilizing substances that are poorly soluble in water, it is used, for example, to solubilize poorly soluble polysaccharides (Non-Patent Document 1). [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Japanese Patent Application Publication No. 8-508637 [Non-patent literature]

[0012] [Non-Patent Document 1] Journal of Applied Polymer Science, 123, 3772-3780 (2011). Summary of the Invention [Problem to be solved by the invention]

[0013] In order to diagnose and treat diseases using microRNA, a method for detecting microRNA, which is present in very small amounts and is easily degraded, without reducing the detectable amount is required. The present invention meets this need by providing a method for detecting microRNA from a biological sample containing microRNA without reducing the detectable amount even after long-term storage.

[0014] Furthermore, glycine hydrochloride is a crystalline solid at room temperature. When used for the above-mentioned purposes, it is often used in the form of an aqueous solution. An aqueous solution of glycine hydrochloride is prepared by dissolving solid (crystalline) glycine hydrochloride in water (distilled water, etc.), but complete dissolution requires a relatively long period of time and thorough stirring. Furthermore, glycine hydrochloride is an acidic substance that irritates the skin and mucous membranes. Therefore, it is necessary to prevent scattering in the solid (powdered) state and splashing in the aqueous solution state, and therefore, safety precautions are required for handling, particularly when transporting the composition. The present invention provides a composition that can be used easily and safely in the above-mentioned microRNA detection method. [Means for solving the problem]

[0015] To verify whether it is possible to detect microRNAs equivalent to those detected before storage in biological samples stored for long periods of time by methods other than freezing, the inventors first compared the amount of microRNA detected in biological samples stored for long periods of time without freezing before and after storage.

[0016] For the purpose of disease diagnosis and treatment, quantitative values ​​of specific microRNAs or values ​​calculated from a combination of these quantitative values ​​are used for diagnosis and treatment. In this case, for example, if the detected amount of each microRNA decreases to less than 70%, significant problems can occur in diagnosing and treating the disease, but even a decrease to less than 90% can cause problems. Because the combination of microRNAs to be quantified varies depending on the target disease and treatment, it is not realistic to find conditions under which the fluorescence intensity of all microRNAs does not decrease. Therefore, instead of measuring the change in the detected amount of each microRNA, a method is known in which the change in the detected amount of all microRNAs arranged on a microarray is estimated by summing the fluorescence intensities of all microRNAs detected on the microarray. The inventors used the sum of the fluorescence intensities of all microRNAs on the microarray as the detected amount of microRNA and set 70% of the detected amount before storage of the biological sample as the threshold for use in diagnosing and treating the disease.

[0017] As described below in Comparative Example 4, after adjusting the serum, it was stored at 23°C for 48 hours, and then microRNA was detected. As a result, the amount of detected microRNA decreased to 28.9% of that before storage (Comparative Example 5). In other words, it was found that in biological samples stored for a long period without freezing, the amount of detected microRNA significantly decreased compared to before storage as a result of degradation by RNase.

[0018] As a result of extensive research to overcome the above-mentioned problems, the inventors have found that by mixing an acid with a biological sample, storing it for a predetermined period of time, and then recovering the microRNA, it is possible to detect microRNA at levels equivalent to or greater than those detected before storage.

[0019] Furthermore, in order to solve the problems of dissolution rate and safety of glycine hydrochloride described above, the inventors conducted extensive research to find a composition of neutral amino acid hydrochloride with higher solubility. As a result, they discovered a homogeneous semi-solid composition consisting of a neutral amino acid hydrochloride, water, and an alcohol, and found that this composition has improved water solubility compared to conventional solid (crystalline) neutral amino acid hydrochlorides.

[0020] That is, the present invention provides the following (1) to (18). (1) A method for detecting microRNA in a biological sample, comprising: (A) mixing a biological sample with an acid to obtain a biological sample mixture; (B) storing the biological sample mixture obtained in step (A); (D) recovering microRNA from the biological sample mixture obtained by step (B); (E) detecting the microRNA recovered by step (D); A detection method comprising: (2) The detection method according to (1), wherein in step (A), the pH of the obtained biological sample mixture is 2.0 to 4.0. (3) The detection method according to (1) or (2), which comprises, prior to step (D), step (C) of mixing a base with the biological sample mixture obtained in step (B) to adjust the pH to 6.0 to 9.0. (4) The detection method according to (3), wherein in step (C), the pH is adjusted to 7.5 to 9.0. (5) The method according to any one of (1) to (4), wherein the biological sample is blood, serum, or plasma. (6) The detection method according to any one of (1) to (5), wherein the acid used in step (A) is glycine hydrochloride, alanine hydrochloride, citric acid, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, or oxalic acid. (7) The detection method according to any one of (1) to (5), wherein the acid used in step (A) is a homogeneous composition consisting of a hydrochloride salt of a neutral amino acid, water, and an alcohol, and is a semi-solid composition at 23°C. (8) The detection method according to any one of (1) to (7), wherein in step (B), the biological sample mixture is stored for 96 hours or less. (9) The detection method according to any one of (1) to (8), wherein in step (B), the biological sample mixture is stored at a temperature of 1 to 30°C. (10) The detection method according to (3) or (4), wherein the base used in step (C) is trishydroxymethylaminomethane, N-[tris(hydroxymethyl)methyl]glycine, triethanolamine, sodium hydroxide, potassium hydroxide, calcium hydroxide, N,N-bis(2-hydroxyethyl)glycine, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid, or 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid. (11) The detection method according to any one of (1) to (10), wherein in step (E), the method for detecting microRNA is a microarray method. (12) The detection method according to any one of (1) to (11), wherein the microRNA is a microRNA used as a disease marker. (13) The detection method according to (12), wherein the disease is cancer or dementia. (14) A homogeneous composition comprising a hydrochloride salt of a neutral amino acid, water, and an alcohol, which is used as an acid in step (A) of the detection method described in (1), and which is semi-solid at 23°C. (15) The composition according to (14), wherein the weight ratio of the neutral amino acid hydrochloride in the composition is 65 wt% or more and 70 wt% or less, and the weight ratio of the alcohol in the composition is 2 wt% or more and 6 wt% or less. (16) The composition according to (14) or (15), wherein the hydrochloride of the neutral amino acid is glycine hydrochloride or alanine hydrochloride. (17) The composition according to any one of (14) to (16), wherein the alcohol is methanol or ethanol. (18) A container for storing a biological sample, in which the composition according to any one of (14) to (17) is enclosed. [Effects of the Invention]

[0021] According to the present invention, microRNA can be detected without a decrease in the amount of detection even after a biological sample containing microRNA has been collected and stored for a long period of time, for example, not under frozen conditions.

[0022] Furthermore, the composition of the present invention can be dissolved in water more quickly than a solid neutral amino acid hydrochloride. Furthermore, because it is a homogeneous semi-solid composition, there are no safety concerns about scattering of the solid (powdered) state or splashing of the aqueous solution, which are issues with solid and aqueous solutions of neutral amino acid hydrochlorides, and the composition is easy to handle, especially when transporting. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides a method for detecting microRNA in a biological sample, comprising the following steps: (A) mixing a biological sample with an acid to obtain a biological sample mixture; (B) storing the biological sample mixture obtained in step (A) for a predetermined period of time; (D) recovering microRNA from the biological sample mixture obtained by step (B); (E) A step of detecting the microRNA recovered in step (D). The present invention will be described below for each of the above steps.

[0024] Step (A) of the present invention is a step of mixing an acid to obtain a biological sample mixture. Preferably, adjusting the pH of the biological sample to 2.0 to 4.0, more preferably 2.5 to 4.0, reduces the activity of RNase in the biological sample and inhibits the degradation of microRNA in the biological sample. Adjusting the pH of the biological sample to 2.0 or higher is preferred because it prevents the cleavage of the phosphodiester bond of microRNA by acid hydrolysis. Adjusting the pH of the biological sample to 4.0 or lower is also preferred because it sufficiently reduces the enzymatic activity of RNase contained in the biological sample.

[0025] The biological sample used in the present invention refers to a sample obtained from a living organism that contains microRNA. Specific examples include serum, plasma, whole blood, bone marrow fluid, lymph, saliva, bile, pancreatic juice, ascites, body fluids, and secretions. Other examples include tissues obtained from a living organism and cultures of cultured cells.

[0026] Furthermore, the biological sample is not particularly limited as long as it is collected from an organism, but is preferably derived from a human. The biological sample used in the present invention is preferably blood, serum, or plasma derived from a human, and more preferably serum or plasma derived from a human.

[0027] Methods for collecting biological samples are well known in the art, for example, blood may be drawn by a phlebotomist, such as a skilled medical professional, using a blood collection needle, followed by centrifugation to obtain serum or plasma.

[0028] The acid to be mixed with the biological sample is an acid that can be mixed with the biological sample in this step. Examples of acids used in the present invention include glycine hydrochloride, alanine hydrochloride, citric acid, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, and oxalic acid. Glycine hydrochloride or citric acid is particularly preferred. Also preferred is a homogeneous composition consisting of a neutral amino acid hydrochloride, water, and an alcohol, which is semi-solid at 23°C.

[0029] The method for mixing the biological sample with the acid is not particularly limited. For example, the biological sample may be placed in a container containing the acid and mixed by pipetting using a micropipette or the like. Alternatively, the biological sample may be placed in a sealable container containing the acid, sealed, and then mixed by placing the container in a shaker such as a vortex mixer, or by inverting the container. In particular, the method of mixing by inverting the container allows for rapid mixing even in an environment without special equipment, such as a medical examination vehicle used for regular medical examinations.

[0030] Step (B) of the present invention is a step of storing the biological sample mixture for a predetermined time period, which is the time period during which the acid mixture can exert its inhibitory effect on the degradation of microRNA in the biological sample, and can be appropriately determined taking into account the time period leading up to step (D).

[0031] For example, if the operation is temporarily suspended after step (A), the biological sample mixture obtained in step (A) is stored, and then microRNA recovery and detection operations are performed, this storage time can be the predetermined time for that step. Also, for example, if the biological sample mixture obtained in step (A) is temporarily stored and then transported, the total time of this storage time and transportation time can be the predetermined time for that step.

[0032] More specifically, for example, in the case of a regular health checkup, the location where the blood collector obtains the biological sample is separate from the location where the microRNA recovery and detection operations are performed, and the biological sample mixture is stored in a sealable container at the location where it is obtained and then transported, the storage time and transportation time can be the specified time for the process.

[0033] Specifically, the specified time is preferably 1 hour or more, as this is the time during which the effects of the present invention can be obtained. In particular, when actually used, taking into consideration that the location where the blood collector obtains the biological sample may be separated from the location where the microRNA recovery and detection operations are performed, 24 hours or more is more preferable, and 48 hours or more is even more preferable.

[0034] On the other hand, the time period during which the effects of the present invention can be obtained may vary depending on the temperature and the type of biological sample, but the predetermined time period is preferably within approximately 96 hours.

[0035] The biological sample mixture in step B can be stored under the temperature conditions typically used for collection, transportation, etc. of biological samples, without the need for refrigeration. Specifically, the temperature is 1 to 30°C, preferably 15 to 25°C. In particular, in the detection of microRNA in step (E), storage conditions that allow 70% or more of the amount of microRNA detected by, for example, a microarray method are preferred. More preferably, storage conditions that allow 90% or more of the amount of microRNA detected are preferred.

[0036] In this process, it is preferable to add, prior to step (D), step (C) of adjusting the pH by mixing a base with the biological sample mixture. The pH adjusted in step (C) is preferably 6.0 to 9.0, which allows for the detection of microRNAs equivalent to those before storage. A preferred pH is 7.5 to 9.0, more preferably 7.5 to 8.5, which allows for the detection of microRNAs equivalent to or greater than those before storage. If the pH of the biological sample mixture is adjusted to less than 6.0 or more than 9.0, the amount of microRNA detected may be lower than that before storage.

[0037] The base mixed in step (C) is a base capable of adjusting the pH of the biological sample mixture to 6.0 to 9.0 when mixed with the biological sample mixture. Examples include tris(hydroxymethyl)aminomethane (Tris), N-[tris(hydroxymethyl)methyl]glycine (tricine), triethanolamine, sodium hydroxide, potassium hydroxide, calcium hydroxide, N,N-bis(2-hydroxyethyl)glycine (bicine), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid (TAPSO). Tris(hydroxymethyl)aminomethane is particularly preferred as the base used in the present invention.

[0038] The method for mixing the biological sample mixture with the base is not particularly limited. For example, the base may be added to a container that has already contained the biological sample mixture, and the mixture may be mixed by pipetting using a micropipette or the like. Alternatively, the base may be added to a sealable container that has already contained the biological sample mixture, and the container may be sealed, and then the container may be placed in a shaker such as a vortex mixer to mix the mixture, or the container may be turned upside down to mix the mixture.

[0039] Step (D) of the present invention is a step of extracting and recovering microRNA. The recovery method used can be any method known in the technical field of nucleic acid recovery. Examples include phenol-chloroform extraction, column chromatography, ethanol precipitation, and magnetic bead adsorption.

[0040] Step (E) of the present invention is a step of detecting the collected microRNA. The detection method used can be a method well known in the technical field of nucleic acid detection. Examples include microarray, PCR, Northern blotting, and sequence analysis. The microarray method is particularly preferred as the method used in the present invention.

[0041] An example of the microRNA of the present invention is a microRNA used as a disease marker. The present invention can provide information for diagnosing a disease by providing a detection value of the expression level of the microRNA that serves as a disease marker in a sample. An example of the disease marker of the present invention is a marker for cancer or dementia.

[0042] The present invention also relates to a composition comprising a hydrochloride salt of a neutral amino acid, water, and an alcohol.

[0043] The composition of the present invention is characterized by being a homogeneous semi-solid at 23°C, unlike a solid (crystalline) neutral amino acid hydrochloride, an aqueous solution of a neutral amino acid hydrochloride, or a suspension in which a solid neutral amino acid hydrochloride is dispersed.

[0044] The acid used in step (A) of the present invention is a homogeneous composition consisting of a hydrochloride of a neutral amino acid, water, and an alcohol, and is semi-solid at 23°C. This composition is preferred because it has the advantage of quickly dissolving in the biological sample mixture and being less likely to scatter. "Semi-solid" refers to a state in which a substance is easily deformed by the application of force and retains its deformed shape even after the force is removed. Neutral amino acid hydrochloride (crystal) is a solid that is not easily deformed by force, so it does not fall under this definition. Furthermore, a suspension of neutral amino acid hydrochloride does not retain its deformed shape, so it does not fall under this definition.

[0045] A composition can be judged to be semi-solid by determining whether it can maintain its shape after being deformed by applying force. Specifically, a composition is judged to be semi-solid if it can maintain a height of 0.8 cm or more for 10 minutes at 23°C after being deformed into a cube with sides of 0.5 cm and a height of 1.0 cm.

[0046] The composition of the present invention is characterized by its rapid dissolution in water at room temperature without the need for stirring or other procedures, as compared with solid neutral amino acid hydrochlorides.

[0047] The rapid dissolution of a composition in water can be evaluated by adding the composition to water and observing the dissolution. Distilled water in an amount 10 times the weight of the composition is added to a container containing the composition, and after standing for a certain period of time, if no solid residue is observed in the water, it is determined that the composition has rapidly dissolved. If solid residue is observed in the water, it is determined that the composition has not dissolved. Specifically, 1 mL of distilled water is added to a 2 mL PP tube containing 0.1 g of the composition, and after standing for 1 minute at 23°C, if no solid residue is observed in the aqueous solution, it is determined that the composition has rapidly dissolved. If solid residue is observed in the aqueous solution, it is determined that the composition has not dissolved.

[0048] When the solid (crystal) of glycine hydrochloride was evaluated by the above method, it was determined that it was not dissolved because the solid remained in the aqueous solution (see Comparative Example 11).

[0049] The composition of the present invention contains a neutral amino acid hydrochloride, water, and an alcohol, and is a semi-solid composition at 23°C.

[0050] The composition of the present invention preferably contains a neutral amino acid hydrochloride in a weight ratio of 65 wt% or more and 70 wt% or less, an alcohol in a weight ratio of 2 wt% or more and 6 wt% or less, and water. The weight ratio of the neutral amino acid hydrochloride and the alcohol in the composition can be calculated by diluting the composition with water and analyzing it by gas chromatography. The weight ratio of water in the composition can be calculated by vaporizing the water in the composition by heating using a moisture vaporizer and then measuring the amount of vaporized water using a Karl Fischer moisture meter.

[0051] The neutral amino acid hydrochloride used in the composition of the present invention includes the hydrochloride of the amino acid that has the same number of acidic carboxyl groups and basic amino groups in the molecule.For example, the amino acids that constitute protein include, but are not limited to, hydrochlorides of glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, asparagine, glutamine, proline, phenylalanine, tyrosine, tryptophan, etc.From the viewpoint of cost and availability, glycine hydrochloride or alanine hydrochloride is preferred.

[0052] The alcohol used in the composition of the present invention is preferably, for example, an alcohol having 1 to 8 carbon atoms. Furthermore, the alcohol of the present invention is preferably a monohydric alcohol to a trihydric alcohol. Specific examples include monohydric alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, i-butanol, t-butanol, 3-methyl-1-butanol, 1-pentanol, 2-pentanol, n-hexanol, cyclohexanol, 1-heptanol, 1-octanol, 2-octanol, 2-methoxyethanol, allyl alcohol, furfuryl alcohol, and phenol, and polyhydric alcohols such as ethylene glycol, propylene glycol, butylene glycol, and glycerin. From the viewpoints of cost and availability, however, methanol or ethanol is preferred.

[0053] In the case of a composition (aqueous solution) consisting only of glycine hydrochloride and water, if the weight ratio of glycine hydrochloride exceeds 50 wt%, the glycine hydrochloride does not dissolve in water at 23° C., forming a suspension in which solid glycine hydrochloride is dispersed in water. Similarly, in the case of a composition (aqueous solution) consisting only of alanine hydrochloride and water, if the weight ratio of alanine hydrochloride exceeds 50 wt%, the alanine hydrochloride does not dissolve in water at 23° C., forming a suspension in which solid alanine hydrochloride is dispersed in water. The composition of the present invention contains an alcohol in addition to a neutral amino acid hydrochloride and water, and as a result, is obtained as a homogeneous semisolid composition rather than a suspension.

[0054] The composition of the present invention can be dissolved in water to easily prepare an aqueous solution of a neutral amino acid hydrochloride salt at a desired concentration. It can also be dissolved in an aqueous solvent. Examples of aqueous solvents include buffer solutions, culture media, and liquid biological samples. Examples of liquid biological samples include blood, serum, plasma, urine, saliva, and tears.

[0055] The composition of the present invention can be dissolved in a solvent by adding the composition to the solvent or by adding the solvent to a container in which the composition has been sealed in advance. The container in which the composition has been sealed in advance is not particularly limited, but examples thereof include a plastic tube and a glass tube.

[0056] A container pre-filled with the composition of the present invention can be used to store biological samples, and has the advantages of preventing the composition from scattering when opened and of rapidly stabilizing the biological sample by acidification when the biological sample is added.

[0057] The amount of the composition of the present invention dissolved in the solvent may be adjusted appropriately depending on the desired concentration, but is preferably 0.01 g to 10.0 g, more preferably 0.1 g to 1.0 g, of the composition per 100 mL of solvent.

[0058] Since a solution of the composition of the present invention is acidic, the composition of the present invention can be used as a pH adjuster in the same manner as conventional hydrochlorides of neutral amino acids.

[0059] For example, in the case of a composition of the present invention consisting of glycine hydrochloride, water, and methanol or ethanol, Step (1): mixing glycine hydrochloride, water, and methanol or ethanol in a predetermined weight ratio to obtain a suspension of glycine hydrochloride; Step (2): Heating the suspension obtained in step (1) to dissolve glycine hydrochloride; Step (3): A step of cooling the solution of glycine hydrochloride obtained in step (2) to obtain a composition; Here, the weight ratio of glycine hydrochloride mixed in step (1) is 65 wt % or more and 70 wt % or less, and the weight ratio of ethanol or methanol is 2 wt % or more and 6 wt % or less.

[0060] When preparing by the above method, in step (1), glycine hydrochloride does not dissolve and a suspension of glycine hydrochloride solids is formed in water. However, in step (2), heating dissolves glycine hydrochloride to form a homogeneous solution, and in step (3), cooling yields the desired homogeneous semisolid composition.

[0061] The method for mixing glycine hydrochloride, water, and ethanol or methanol in step (1) is not particularly limited, and may be, for example, a method in which the mixture is mixed using a vortex mixer at 23°C for 1 minute.

[0062] In step (2), the suspension is heated to a temperature that is equal to or lower than the boiling point of the component with the lowest boiling point and that can dissolve glycine hydrochloride. For example, when ethanol (boiling point 78.4°C) is used, heating is performed at 75°C, and when methanol (boiling point 64.7°C) is used, heating is performed at 60°C. The heating method is not particularly limited, but a water bath or block incubator may be used. The heating time is preferably 5 minutes or more and 30 minutes or less. During heating, heating is preferably performed in a sealed state to prevent evaporation of water and ethanol or methanol.

[0063] In step (2), whether glycine hydrochloride has dissolved can be determined by visual observation. If no solid remains in the aqueous solution after heating, it is determined to have dissolved, and if solid remains in the aqueous solution, it is determined to have not dissolved.

[0064] In step (3), the aqueous solution may be cooled using a commonly used cooling device, or the aqueous solution after heating may be left to cool in the heated container or transferred to another container. The cooling time is preferably 10 minutes or more and 60 minutes or less. The temperature after cooling is preferably 20 to 25°C.

[0065] The composition of the present invention can also be prepared from alanine hydrochloride using the same process as for glycine hydrochloride.

[0066] The composition of the present invention can also be prepared using hydrochlorides of neutral amino acids other than glycine hydrochloride and alanine hydrochloride in the same manner. [Example]

[0067] Example 1 Detection of microRNA from serum preserved after mixing with glycine hydrochloride 1 (A) A step of mixing an acid with a biological sample Blood was collected from healthy subject X and serum was prepared. 300 μL of serum immediately after preparation was mixed with 90 μL of 1 M glycine hydrochloride solution (Fujifilm Wako Pure Chemical Industries, Ltd.). The serum pH was measured using a handheld pH meter (HORIBA, Ltd.), and it was confirmed that the pH had decreased from 7.5 to 2.8 due to the addition of acid.

[0068] (B) A step of storing the biological sample mixture for a predetermined period of time The serum mixed with the acid in step (A) was left to stand at 23°C for 48 hours.

[0069] (C) A step of mixing a base into the biological sample mixture The serum stored in step (B) was mixed with 27 μL of a 4 M aqueous solution of trishydroxymethylaminomethane (Nacalai Tesque, Inc.). Using a handheld pH meter, it was confirmed that the pH had increased from 2.8 to 7.5 due to the addition of the base.

[0070] (D) Step of recovering microRNA from the biological sample mixture MicroRNA was extracted from the serum mixed with the base in step (C) using the RNA extraction reagent from 3D-Gene (registered trademark) RNA extraction reagent from liquid sample kit (Toray Industries, Inc.) according to the protocol specified by the company.

[0071] (E) Step of detecting microRNA The microRNAs collected from serum in step (D) were fluorescently labeled using the 3D-Gene® miRNA Labeling kit (Toray Industries, Inc.) according to the company's protocol. Hybridization was performed using the 3D-Gene® Human miRNA Oligo chip (Toray Industries, Inc.) DNA microarray chip, equipped with probes with sequences complementary to 2,565 types of microRNAs, according to the company's protocol. The DNA microarray chip was scanned using a 3D-Gene® Scanner (Toray Industries, Inc.) to capture images. Fluorescence intensity was quantified using 3D-Gene® Extraction (Toray Industries, Inc.), and the sum of the fluorescence intensities of microRNAs detected above the detection limit was calculated to determine the amount of microRNA detected. Table 1 shows the amount of microRNA detected.

[0072] Examples 2 to 5: Detection of microRNA from serum preserved after mixing with glycine hydrochloride 2 When mixing the base, In Example 2, the pH was adjusted to 7.7 by adding 30 μL of a 4 M aqueous solution of tris(hydroxymethyl)aminomethane. In Example 3, the pH was adjusted to 8.0 by adding 36 μL of a 4 M aqueous solution of tris(hydroxymethyl)aminomethane. In Example 4, the pH was adjusted to 8.5 by adding 60 μL of a 4 M aqueous solution of tris(hydroxymethyl)aminomethane. In Example 5, the pH was adjusted to 9.0 by adding 120 μL of a 4 M aqueous solution of tris(hydroxymethyl)aminomethane. Except for these adjustments, the same procedures as in Example 1 were carried out. Table 1 shows the amounts of microRNA detected in each of Examples 2 to 5.

[0073] Examples 6 and 7: Detection of microRNA from serum preserved after mixing with glycine hydrochloride 3 When mixing the base, In Example 6, the pH was adjusted to 6.0 by adding 21 μL of a 4 M aqueous solution of tris(hydroxymethyl)aminomethane. In Example 7, the pH was adjusted to 7.0 by adding 22.5 μL of a 4 M aqueous solution of tris(hydroxymethyl)aminomethane. Except for these adjustments, the same procedures as in Example 1 were carried out. Table 1 shows the amounts of microRNA detected in Examples 6 and 7.

[0074] Example 8: Detection of microRNA from serum preserved after mixing with glycine hydrochloride without mixing with base In Example 8, except that no base was mixed, the same method as in Example 1 was used. Table 1 shows the amount of microRNA detected in Example 8.

[0075] Comparative Example 1: Detection of microRNA from serum stored without adding acid, without adding base Comparative Example 1 was carried out in the same manner as in Example 1, except that the acid and base were not mixed. Tables 1 and 2 show the amounts of microRNA detected in Comparative Example 1.

[0076] Comparative Example 2: Detection of microRNA from freshly prepared serum 1 MicroRNA was recovered from 300 μL of serum collected from healthy subject X in the same manner as in step (D) of Example 1, without performing steps (A) to (C) of Example 1, and the recovered microRNA was detected in the same manner as in step (E) of Example 1. Tables 1 and 2 show the detected amounts of microRNA.

[0077] [Table 1]

[0078] When serum was mixed with acid to adjust the pH to 2.8, stored at 23°C for 48 hours, and then mixed with base to adjust the pH to 7.5-8.5 (Examples 1-4), the same or greater (100% or greater) levels of microRNA were detected compared to when freshly prepared serum was used (Comparative Example 2). When base was mixed and the pH was adjusted to 9.0 (Example 5), 90% or greater levels of microRNA were detected compared to when freshly prepared serum was used (Comparative Example 2). Furthermore, when base was mixed and the pH was adjusted to 6.0 and 7.0 (Examples 6 and 7), the same or greater levels of microRNA were detected compared to when freshly prepared serum was used (Comparative Example 2). Furthermore, when base was not mixed (Example 8), the same or greater levels of microRNA were detected compared to when freshly prepared serum was used (Comparative Example 2).

[0079] On the other hand, when serum was stored at 23°C for 48 hours without adding acid (Comparative Example 1), the amount of microRNA detected decreased to less than 30% compared to when freshly prepared serum was used (Comparative Example 2). This is thought to be due to the microRNA being decomposed during storage because no acid was added.

[0080] These results demonstrate that storing serum after adding acid is important for detecting microRNAs, and that adding a base after storage to adjust the pH to 7.5-9.0 is particularly important for detecting microRNAs.

[0081] Example 9: Detection of microRNA from serum preserved after mixing with glycine hydrochloride 4 The same procedure as in Example 1 was used, except that the pH was adjusted to 4.0 by adding 45 μL of a 1 M glycine hydrochloride solution when mixing the acid, and the pH was adjusted to 7.7 by adding 10 μL of a 4 M trishydroxymethylaminomethane solution when mixing the base. Table 2 shows the amount of microRNA detected.

[0082] Comparative Example 3: Detection of microRNA from serum preserved without adding acid Comparative Example 3 was carried out in the same manner as Example 1, except that no acid was added and the pH was adjusted to 7.7 by adding 0.75 μL of 4 M aqueous trishydroxymethylaminomethane solution when the base was added. Table 2 shows the amount of microRNA detected.

[0083] [Table 2]

[0084] When serum was mixed with acid to adjust the pH to 4.0, stored at 23°C for 48 hours, and then mixed with base to adjust the pH to 7.7 (Example 9), the same or higher (90% or more) amount of microRNA was detected compared to when serum was used immediately after preparation (Comparative Example 2).

[0085] On the other hand, when serum was stored at 23°C for 48 hours without adding acid, and then a base was added to adjust the pH to 7.7 (Comparative Example 3), and when no base was added (Comparative Example 1), the amount of microRNA detected fell to less than 30% compared to when freshly prepared serum was used (Comparative Example 2). This is thought to be due to the microRNA being decomposed during storage because no acid was added.

[0086] These results demonstrate that mixing acid with serum before storage and adjusting the pH to 4.0 or below is important for detecting microRNAs.

[0087] Examples 10 to 12 Detection of microRNA from serum preserved after mixing with glycine hydrochloride 5 The storage time of the biological sample mixture is In Example 10, 2 hours; In Example 11, 6 hours; In Example 12, 96 hours; The same procedures as in Example 1 were carried out, except that in Examples 10 to 12, the pH was adjusted to 7.7 by adding 30 μL of a 4 M aqueous trishydroxymethylaminomethane solution when mixing the base. Table 3 shows the amount of microRNA detected in each of Examples 10 to 12.

[0088] [Table 3]

[0089] When serum was mixed with acid to adjust the pH to 2.8, then stored at 23°C for 2, 6, and 96 hours, and then base was added and adjusted to pH 7.7 (Examples 10 to 12), the same or greater amount of microRNA (100% or more) was detected compared to when serum was used immediately after preparation (Comparative Example 2).

[0090] From the above, it was revealed that this method can detect microRNAs from serum stored at 23°C for at least 2 to 96 hours.

[0091] Example 13: Detection of microRNA from serum preserved after mixing with citric acid 1 The same procedure as in Example 1 was used, except that serum collected from healthy subject Y was used, the pH was adjusted to 3.1 by adding 90 μL of 1 M citric acid aqueous solution (Sigma-Aldrich Co. LLC.) when mixing the acid, and the pH was adjusted to 7.8 by adding 90 μL of 4 M trishydroxymethylaminomethane aqueous solution when mixing the base. Table 4 shows the amount of microRNA detected.

[0092] Examples 14 and 15: Detection of microRNA from serum preserved after mixing with citric acid 2 When mixing the base, In Example 14, 63 μL of a 4 M aqueous solution of tris(hydroxymethyl)aminomethane was added and the pH was adjusted to 6.0. In Example 15, 67.5 μL of a 4 M aqueous solution of trishydroxymethylaminomethane was added and adjusted to pH 7.0. Other than that, the same method as in Example 13 was used. Table 4 shows the detected amounts of microRNA.

[0093] Example 16: Detection of microRNA from serum preserved after mixing with citrate without mixing with base Except for not mixing the base, the same method as in Example 13 was used. Table 4 shows the amount of microRNA detected.

[0094] Comparative Example 4: Detection of microRNA from freshly prepared serum 2 MicroRNA was recovered from 300 μL of serum collected from healthy subject Y in the same manner as in step (D) of Example 1, without performing steps (A) to (C) of Example 1, and the recovered microRNA was detected in the same manner as in step (E) of Example 1. Table 4 shows the amount of detected microRNA.

[0095] [Table 4]

[0096] When serum was mixed with citric acid, stored at 23°C for 48 hours, and then a base was added to adjust the pH to 7.8 (Example 13), the same or greater amount of microRNA (100% or more) was detected compared to when freshly prepared serum was used (Comparative Example 4). Furthermore, when a base was added after storage to adjust the pH to 6.0 or 7.0 (Examples 14 and 15), and when no base was added (Example 16), the same or greater amount of microRNA (70% or more) was detected compared to when freshly prepared serum was used (Comparative Example 4).

[0097] From the above results, it became clear that the effect can be obtained regardless of the type of acid mixed with serum, whether it is glycine hydrochloride or citric acid.

[0098] Example 17 Detection of microRNA from serum preserved after mixing with glycine hydrochloride 6 The same procedure as in Example 1 was used, except that serum collected from healthy subject Z was used, the pH was adjusted to 2.0 by adding 180 μL of a 1 M glycine hydrochloride solution when mixing the acid, and the pH was adjusted to 7.7 by adding 60 μL of a 4 M trishydroxymethylaminomethane solution when mixing the base. Table 5 shows the amount of microRNA detected.

[0099] Comparative Example 5: Detection of microRNA from freshly prepared serum 3 MicroRNA was recovered from 300 μL of serum collected from healthy subject Z in the same manner as in step (D) of Example 1, without performing steps (A) and (B) of Example 1, and the recovered microRNA was detected in the same manner as in step (E) of Example 1. Tables 5 and 6 show the detected amounts of microRNA.

[0100] [Table 5]

[0101] When serum was mixed with acid to adjust the pH to 2.0, stored at 23°C for 48 hours, and then mixed with base to adjust the pH to 7.7 (Example 17), the same or greater amount of microRNA (more than 100%) was detected compared to when serum was used immediately after preparation (Comparative Example 5).

[0102] These results demonstrate that mixing acid with serum before storage and adjusting the pH to at least 2.0 to 4.0 is important for detecting microRNAs. Example 18: Detection of microRNA from serum preserved after mixing with glycine hydrochloride 7 The same procedure as in Example 1 was used, except that serum collected from healthy subject Z was used, the pH was adjusted to 2.8 by adding composition 2 described in Example 19 below when mixing the acid, and the pH was adjusted to 7.7 by adding 30 μL of a 4 M aqueous trishydroxymethylaminomethane solution when mixing the base. Table 6 shows the amount of microRNA detected.

[0103] [Table 6]

[0104] When serum was mixed with semi-solid composition 2 at 23°C, which was homogeneous and consisted of a neutral amino acid hydrochloride, water, and alcohol, and the pH was adjusted to 2.8, and then stored at 23°C for 48 hours, and then a base was added and the pH was adjusted to 7.7 (Example 18), the same or greater amount of microRNA (100% or more) was detected compared to when serum was used immediately after preparation (Comparative Example 4).

[0105] From the above results, it became clear that the acid mixed with serum before storage can be effective even if it is a homogeneous composition consisting of a neutral amino acid hydrochloride, water, and alcohol, and is a semi-solid composition at 23°C.

[0106] Example 19 Compositions consisting of glycine hydrochloride, water, and ethanol (Compositions 1 to 6) (1) Preparation of the composition To a 2 mL PP tube, 650 mg (65.0 wt%) of glycine hydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.), 20 mg (2.0 wt%) of ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and 330 mg (33.0 wt%) of distilled water were added to a total amount of 1.0 g, and the mixture was mixed using a vortex mixer at 23°C for 1 minute to obtain a suspension. The tube containing the suspension was heated at 75°C for 10 minutes in a block incubator. After heating, the dissolution state of glycine hydrochloride was evaluated by visual observation. No solid residue was observed in the aqueous solution, confirming that glycine hydrochloride had dissolved. The tube was removed from the block incubator and allowed to stand at 23°C for 30 minutes to obtain Composition 1.

[0107] Compositions 2 to 6 were prepared in the same manner as above using glycine hydrochloride, ethanol, and distilled water in the weight ratios shown in Table 7 so that the total amount was 1.0 g.

[0108] In the preparation process of Compositions 2 to 6, it was confirmed by visual observation that glycine hydrochloride had dissolved after heating.

[0109] (2) Shape evaluation Composition 1 obtained in (1) was deformed into a cube with sides of 0.5 cm and a height of 1.0 cm using a spatula on a plastic petri dish. After the deformation, the cube was left to stand at 23°C for 10 minutes, and the height was measured. As a result, the height was 0.8 cm or more, and the deformed shape was maintained, and therefore, composition 1 was determined to be semi-solid.

[0110] The state of compositions 2 to 6 was evaluated in the same manner as above, and as a result, all compositions maintained their shape after deformation, and composition 1 was therefore determined to be in a semi-solid state.

[0111] (3) Evaluation of solubility The solubility of composition 1 obtained in (1) was evaluated. 1 mL of distilled water was added to a 2 mL PP tube containing 0.1 g of composition 1, and the solution was left to stand at 23°C for 1 minute. The state of dissolution was evaluated by visual observation. No solid residue was observed in the aqueous solution, indicating that the composition had dissolved rapidly.

[0112] The solubility of Compositions 2 to 6 was evaluated in the same manner as above, and as a result, no solid residue was observed in the aqueous solution, indicating that the compositions were dissolved quickly.

[0113] Comparative Example 6 Compositions consisting of glycine hydrochloride, water, and ethanol (Compositions 7 to 20) (1) Preparation of the composition Compositions 7 to 20 were prepared in the same manner as in Example 19(1) using glycine hydrochloride, ethanol, and distilled water in the weight ratios shown in Table 7.

[0114] In the preparation process of compositions 7 to 15, it was confirmed by visual observation that glycine hydrochloride had dissolved after heating. On the other hand, in the preparation process of compositions 16 to 20, solids remained in the aqueous solution even after heating, so it was determined that some of the glycine hydrochloride had not dissolved, and further evaluation was not performed.

[0115] (2) Shape evaluation The state of compositions 7 to 15 obtained in (1) was evaluated using the same method as in Example 19(2). As a result, none of the compositions maintained their shape after deformation, and visual observation confirmed that solids were dispersed in the solution. Therefore, they were determined to be suspensions rather than semi-solids.

[0116] (3) Evaluation of solubility The solubility of compositions 7 to 15 obtained in (1) was evaluated in the same manner as in Example 19(3). As a result, solid precipitation was observed in the aqueous solution, and it was determined that they were not soluble.

[0117] The results obtained in Example 19 and Comparative Example 6 are shown in Table 7.

[0118] [Table 7]

[0119] Compositions 1 to 6 of Example 19 were semisolid and quickly dissolved in distilled water. On the other hand, compositions 7 to 15 of Comparative Example 11 were in the form of a suspension in which precipitated glycine hydrochloride solids were dispersed in an aqueous solution, and did not quickly dissolve even when distilled water was added. Furthermore, in compositions 16 to 20 of Comparative Example 6, glycine hydrochloride did not dissolve even when heated.

[0120] From the above results, it was revealed that when the weight ratio of glycine hydrochloride in the composition is 65 wt% or more and 70 wt% or less, and the weight ratio of ethanol is 2 wt% or more and 6 wt% or less, the desired composition that is semi-solid and rapidly dissolves in water can be obtained.

[0121] Example 20 Compositions consisting of glycine hydrochloride, water, and methanol (Compositions 21 to 26) (1) Preparation of the composition In Example 19(1), ethanol was changed to methanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and the heating temperature was changed from 75°C to 60°C. Compositions 21 to 26 were prepared using glycine hydrochloride, methanol, and distilled water in the weight amounts listed in Table 8.

[0122] In the preparation process of Compositions 21 to 26, it was confirmed by visual observation that glycine hydrochloride had dissolved after heating.

[0123] (2) Shape evaluation The state of compositions 21 to 26 obtained in (1) was evaluated using a method similar to that of Example 19(2). As a result, all compositions maintained their shape after deformation, and were therefore judged to be semi-solid.

[0124] (3) Evaluation of solubility The solubility of compositions 21 to 26 obtained in (1) was evaluated in the same manner as in Example 19(3). As a result, no solid precipitate was observed in the aqueous solution, and it was therefore determined that the compositions were dissolved.

[0125] Comparative Example 7 Compositions consisting of glycine hydrochloride, water, and methanol (Compositions 27 to 40) (1) Preparation of the composition Compositions 27 to 40 were prepared in the same manner as in Example 20(1) using glycine hydrochloride, methanol, and distilled water in the amounts by weight shown in Table 8.

[0126] In the preparation process of compositions 27 to 35, it was confirmed by visual observation that glycine hydrochloride had dissolved after heating. On the other hand, in the preparation process of compositions 36 to 40, solids remained in the aqueous solution even after heating, so it was determined that glycine hydrochloride had not dissolved, and subsequent operations were not carried out.

[0127] (2) Shape evaluation The state of compositions 27 to 35 obtained in (1) was evaluated using a method similar to that of Example 19(2). As a result, none of the compositions maintained their shape after deformation, and visual observation confirmed that solids were dispersed in the solution. Therefore, they were determined to be suspensions rather than semi-solids.

[0128] (3) Evaluation of solubility The solubility of compositions 27 to 35 obtained in (1) was evaluated in the same manner as in Example 19(3). As a result, solid precipitation was observed in the aqueous solution, and it was determined that they were not dissolved.

[0129] The results obtained in Example 20 and Comparative Example 7 are shown in Table 8.

[0130] [Table 8]

[0131] Compositions 21 to 26 of Example 20 were semisolid and quickly dissolved in distilled water. On the other hand, compositions 27 to 35 of Comparative Example 7 were in the form of a suspension in which precipitated glycine hydrochloride solids were dispersed in an aqueous solution, and did not quickly dissolve even when distilled water was further added. Furthermore, in compositions 36 to 40 of Comparative Example 7, glycine hydrochloride did not dissolve even when heated.

[0132] From the above results, it was revealed that, similarly to the case of ethanol, in the case of methanol, when the weight ratio of glycine hydrochloride in the composition is 65 wt % or more and 70 wt % or less and the weight ratio of methanol is 2 wt % or more and 6 wt % or less, the desired composition that is semi-solid and rapidly dissolves in water can be obtained.

[0133] Comparative Example 8 Preparation and evaluation of compositions consisting of glycine hydrochloride, water, and acetone (compositions 41-52) (1) Preparation of the composition In Example 19(1), ethanol was changed to acetone (Fujifilm Wako Pure Chemical Industries, Ltd.), and the heating temperature was changed from 75°C to 60°C. Compositions 41 to 52 were prepared using glycine hydrochloride, acetone, and distilled water in the weight amounts listed in Table 9.

[0134] In the preparation process of compositions 41 to 49, it was confirmed by visual observation that glycine hydrochloride had dissolved after heating. On the other hand, in the preparation process of compositions 50 to 52, solids remained in the aqueous solution even after heating, so it was determined that glycine hydrochloride had not dissolved, and the subsequent operations were not carried out.

[0135] (2) Shape evaluation The state of compositions 41 to 49 obtained in (1) was evaluated using a method similar to that of Example 19(2). As a result, none of the compositions maintained their shape after deformation, and visual observation confirmed that solids were dispersed in the solution. Therefore, they were determined to be suspensions rather than semi-solids.

[0136] (3) Evaluation of solubility The solubility of compositions 41 to 49 obtained in (1) was evaluated in the same manner as in Example 19(3). As a result, solid precipitation was observed in the aqueous solution, and it was determined that they were not dissolved.

[0137] The results obtained in Comparative Example 8 are shown in Table 9.

[0138] [Table 9]

[0139] When acetone was used instead of ethanol or methanol, the resulting composition was in the form of a suspension in which solid glycine hydrochloride was dispersed in an aqueous solution, and the composition intended by the present invention was not obtained.

[0140] Comparative Example 9 Compositions consisting of glycine hydrochloride, water, and acetonitrile (Compositions 53-64) (1) Preparation of the composition In Example 19(1), ethanol was changed to acetonitrile (Fujifilm Wako Pure Chemical Industries, Ltd.), and compositions 53 to 64 were prepared using glycine hydrochloride, acetonitrile, and distilled water in the weight amounts shown in Table 10.

[0141] In the preparation process of compositions 53 to 61, it was confirmed by visual observation that glycine hydrochloride had dissolved after heating. On the other hand, in the preparation process of compositions 62 to 64, solids remained in the aqueous solution even after heating, so it was determined that glycine hydrochloride had not dissolved, and subsequent operations were not carried out.

[0142] (2) Shape evaluation The state of compositions 53 to 61 obtained in (1) was evaluated using a method similar to that of Example 19(2). As a result, none of the compositions maintained their shape after deformation, and visual observation confirmed that solids were dispersed in the solution. Therefore, they were determined to be suspensions rather than semi-solids.

[0143] (3) Evaluation of solubility The solubility of compositions 53 to 61 obtained in (1) was evaluated in the same manner as in Example 19(3). As a result, solid precipitation was observed in the aqueous solution, and it was determined that they were not dissolved.

[0144] The results obtained in Comparative Example 9 are shown in Table 10.

[0145] [Table 10]

[0146] When acetonitrile was used, the resulting composition was in the form of a suspension in which solid glycine hydrochloride was dispersed in an aqueous solution, and the composition intended by the present invention was not obtained.

[0147] Comparative Example 10 Compositions consisting of glycine hydrochloride and water (Compositions 65 to 68) (1) Preparation of the composition Compositions 65 to 68 were prepared using glycine hydrochloride and distilled water in the weight ratios shown in Table 11, in the same manner as in Example 19(1), except that ethanol was not used.

[0148] In the preparation process of compositions 65 to 67, it was confirmed by visual observation that glycine hydrochloride had dissolved after heating. On the other hand, in the preparation process of composition 68, solids remained in the aqueous solution even after heating, so it was determined that glycine hydrochloride had not dissolved, and subsequent operations were not carried out.

[0149] (2) Shape evaluation The state of compositions 65 to 67 obtained in (1) was evaluated using a method similar to that of Example 19(2). As a result, none of the compositions maintained their shape after deformation, and visual observation confirmed that solids were dispersed in the solution. Therefore, they were determined to be suspensions rather than semi-solids.

[0150] (3) Evaluation of solubility The solubility of compositions 65 to 67 obtained in (1) was evaluated in the same manner as in Example 19(3). As a result, solid precipitation was observed in the aqueous solution, and it was determined that they were not soluble.

[0151] The results obtained in Comparative Example 10 are shown in Table 11.

[0152] [Table 11]

[0153] When only glycine hydrochloride and water were used, the resulting composition was in the form of a suspension in which solid glycine hydrochloride was dispersed in an aqueous solution, and the composition intended by the present invention was not obtained.

[0154] From the results obtained in Comparative Examples 8 to 10, it became clear that the semi-solid composition that is the object of the present invention and that dissolves rapidly in water requires ethanol or methanol in addition to glycine hydrochloride and water.

[0155] Comparative Example 11 Evaluation of the solid solubility of glycine hydrochloride. 1 mL of distilled water was added to a 2 mL PP tube containing 0.1 g of glycine hydrochloride, and the tube was left to stand at 23°C for 1 minute. The state of dissolution was evaluated by visual observation. As solids remained in the aqueous solution, it was determined that the substance had not dissolved.

[0156] Example 21 Serum pH adjustment using compositions consisting of glycine hydrochloride, water, and ethanol (compositions 1 to 6) 1 mL of serum prepared from blood collected from a healthy human volunteer was added to a 2 mL PP tube containing 0.1 g of Composition 1 prepared in Example 19(1), and after standing at 23°C for 1 minute, the dissolution state was evaluated by visual observation. No solids were observed in the serum, and it was determined that each composition had dissolved rapidly. 0.1 mL of the serum was recovered from the upper layer in the tube, and the pH was measured using a handheld pH meter (HORIBA, Ltd.). The pH was 2.7, a significant decrease compared to the pH (7.7) of the serum before addition.

[0157] Using the same method as above, 1 mL of serum was added to tubes containing 0.1 g of compositions 2 to 6, and the state of dissolution was evaluated visually. No solids were found in the serum, and it was determined that compositions 2 to 6 had quickly dissolved. When the pH was measured after standing, the tubes containing compositions 2 and 3 had a pH of 2.7, and the tubes containing compositions 4 to 6 had a pH of 2.6, which was significantly lower than the pH of the serum before addition (7.7).

[0158] Comparative Example 12 Serum pH control using solid glycine hydrochloride. In the same manner as in Example 21, 1 mL of serum was added to a 2 mL PP tube containing 65 mg of solid (crystalline) glycine hydrochloride, and the state of dissolution was evaluated visually. As a result, solids were observed in the serum, and it was determined that the solid glycine hydrochloride did not dissolve quickly. After standing, the pH of the serum was measured and found to be 4.5, a smaller decrease than in Example 21, in which Compositions 1 to 6 were added.

[0159] The results of Example 21 and Comparative Example 12 are shown in Table 12.

[0160] [Table 12]

[0161] The compositions consisting of glycine hydrochloride, water, and ethanol (Compositions 1 to 6) rapidly dissolved in serum and lowered the serum pH. On the other hand, the solid glycine hydrochloride did not rapidly dissolve in serum when left standing, and was unable to sufficiently lower the serum pH.

[0162] From the above, it has become clear that the composition of the present invention can also be used as a pH adjuster. Example 22 Compositions consisting of alanine hydrochloride, water, and ethanol (Compositions 69 and 70) In Example 19(1), glycine hydrochloride was changed to alanine hydrochloride (Sigma), and compositions 69 and 70 were prepared using alanine hydrochloride, ethanol, and distilled water in the weight amounts shown in Table 13.

[0163] In the preparation process of Compositions 69 and 70, it was confirmed by visual observation that alanine hydrochloride was dissolved after heating.

[0164] (2) Shape evaluation The state of compositions 69 and 70 obtained in (1) was evaluated using a method similar to that of Example 19(2). As a result, both compositions maintained their shape after deformation, and were therefore judged to be semi-solid.

[0165] (3) Evaluation of solubility The solubility of compositions 69 and 70 obtained in (1) was evaluated using a method similar to that of Example 19(3). As a result, no solid precipitate was observed in the aqueous solution, and it was therefore determined that the compositions were dissolved.

[0166] Comparative Example 13 Compositions consisting of alanine hydrochloride, water, and ethanol (Compositions 71 to 80) (1) Preparation of the composition Compositions 71 to 80 were prepared in the same manner as in Example 20(1) using alanine hydrochloride, ethanol, and distilled water in the amounts by weight shown in Table 13.

[0167] In the preparation process of compositions 71 to 77, it was confirmed by visual observation that alanine hydrochloride had dissolved after heating. On the other hand, in the preparation process of compositions 78 to 80, solids remained in the aqueous solution even after heating, so it was determined that alanine hydrochloride had not dissolved, and subsequent operations were not carried out.

[0168] (2) Shape evaluation The state of compositions 71 to 77 obtained in (1) was evaluated using a method similar to that of Example 19(2). As a result, none of the compositions maintained their shape after deformation, and visual observation confirmed that solids were dispersed in the solution. Therefore, they were determined to be suspensions rather than semi-solids.

[0169] (3) Evaluation of solubility The solubility of compositions 71 to 77 obtained in (1) was evaluated in the same manner as in Example 19(3). As a result, solid precipitation was observed in the aqueous solution, and it was determined that they were not soluble.

[0170] The results obtained in Example 22 and Comparative Example 13 are shown in Table 13.

[0171] [Table 13]

[0172] Compositions 69 and 70 of Example 22, which used alanine hydrochloride, were semisolid and quickly dissolved in distilled water. On the other hand, compositions 71 to 77 of Comparative Example 13 were in the form of a suspension in which precipitated alanine hydrochloride solids were dispersed in an aqueous solution, and did not quickly dissolve even when distilled water was added. Furthermore, in compositions 78 to 80 of Comparative Example 13, alanine hydrochloride did not dissolve even when heated.

[0173] From the above results, it was revealed that, similarly to the case of glycine hydrochloride, when the weight ratio of alanine hydrochloride in the composition is 65 wt % or more and 70 wt % or less, and the weight ratio of ethanol is 5 wt %, the desired composition that is semi-solid and rapidly dissolves in water can be obtained.

[0174] Comparative Example 14 Compositions consisting of glutamic acid hydrochloride, water, and ethanol (Compositions 81-92) (1) Preparation of the composition In Example 19(1), glycine hydrochloride was changed to glutamic acid hydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.), and compositions 81 to 92 were prepared using glutamic acid hydrochloride, ethanol, and distilled water in the weight amounts shown in Table 14.

[0175] In the preparation process of compositions 81 to 86, it was confirmed by visual observation that glutamic acid hydrochloride dissolved after heating. On the other hand, in the preparation process of compositions 87 to 92, since solids remained in the aqueous solution even after heating, it was determined that glutamic acid hydrochloride did not dissolve, and the subsequent operations were not carried out.

[0176] (2) Shape evaluation The state of compositions 81 to 86 obtained in (1) was evaluated in the same manner as in Example 19(2). As a result, none of the compositions maintained their shape after deformation, and no solid was observed in the solution by visual observation. Therefore, it was determined that the glutamic acid hydrochloride was not in a semi-solid state, but was a solution in which the glutamic acid hydrochloride was completely dissolved. Since the glutamic acid hydrochloride was dissolved, the solubility evaluation shown in Example 19(3) was not performed.

[0177] The results obtained in Comparative Example 14 are shown in Table 14.

[0178] [Table 14]

[0179] When glutamic acid hydrochloride was used, the resulting composition was in the form of a solution in which glutamic acid hydrochloride was completely dissolved, and the composition intended by the present invention could not be obtained.

[0180] Comparative Example 15 Compositions consisting of lysine monohydrochloride, water, and ethanol (Compositions 93-104) (1) Preparation of the composition In Example 19(1), glycine hydrochloride was changed to lysine monohydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.), and compositions 93 to 104 were prepared using lysine monohydrochloride, ethanol, and distilled water in the weight amounts shown in Table 15.

[0181] In the preparation of compositions 93 to 98, it was confirmed by visual observation that lysine monohydrochloride had dissolved after heating. On the other hand, in the preparation of compositions 99 to 104, solids remained in the aqueous solution even after heating, so it was determined that lysine monohydrochloride had not dissolved, and subsequent operations were not carried out.

[0182] (2) Shape evaluation The state of compositions 93 to 98 obtained in (1) was evaluated in the same manner as in Example 19(2). As a result, none of the compositions maintained their shape after deformation, and no solid was observed in the solution by visual observation. Therefore, it was determined that the compositions were not semisolid, but were solutions in which lysine monohydrochloride was completely dissolved. Since lysine monohydrochloride was dissolved, the solubility evaluation described in Example 19(3) was not performed.

[0183] The results obtained in Comparative Example 15 are shown in Table 15.

[0184] [Table 15]

[0185] When lysine monohydrochloride was used, the composition obtained was in the form of a solution in which lysine monohydrochloride was completely dissolved, and the composition of the present invention was not obtained.

[0186] Comparative Example 16 Compositions 105-116 consisting of lysine dihydrochloride, water, and ethanol (1) Preparation of the composition In Example 19(1), glycine hydrochloride was changed to lysine dihydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.), and compositions 105 to 116 were prepared using lysine dihydrochloride, ethanol, and distilled water in the weight amounts shown in Table 16.

[0187] In the preparation of compositions 105 to 113, it was confirmed by visual observation that lysine dihydrochloride had dissolved after heating. On the other hand, in the preparation of compositions 114 to 116, solids remained in the aqueous solution even after heating, so it was determined that lysine dihydrochloride had not dissolved, and subsequent operations were not carried out.

[0188] (2) Shape evaluation The state of compositions 105 to 113 obtained in (1) was evaluated in the same manner as in Example 19(2). As a result, none of the compositions maintained their shape after deformation, and no solid was observed in the solution by visual observation. Therefore, it was determined that the compositions were not semisolid, but were solutions in which lysine dihydrochloride was completely dissolved. Since lysine dihydrochloride was dissolved, the solubility evaluation described in Example 19(3) was not performed.

[0189] The results obtained in Comparative Example 16 are shown in Table 16.

[0190] [Table 16]

[0191] When lysine dihydrochloride was used, the composition obtained was in the form of a solution in which lysine dihydrochloride was completely dissolved, and the composition of the present invention was not obtained.

Claims

1. 1. A method for detecting microRNA in a biological sample, comprising: (A) mixing an acid with a biological sample to obtain a biological sample mixture; (B) storing the biological sample mixture obtained in step (A); (D) recovering microRNA from the biological sample mixture obtained in step (B); (E) detecting the microRNA recovered by step (D); A detection method comprising:

2. 2. The detection method according to claim 1, wherein the pH of the biological sample mixture obtained in step (A) is 2.0 to 4.

0.

3. The detection method according to claim 1 or 2, comprising, prior to step (D), step (C) of mixing a base with the biological sample mixture obtained in step (B) to adjust the pH to 6.0 to 9.

0.

4. 4. The detection method according to claim 3, wherein in step (C), the pH is adjusted to 7.5 to 9.

0.

5. The detection method according to any one of claims 1 to 4, wherein the biological sample is blood, serum, or plasma.

6. 6. The detection method according to claim 1, wherein the acid used in step (A) is glycine hydrochloride, alanine hydrochloride, citric acid, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, or oxalic acid.

7. The detection method according to any one of claims 1 to 5, wherein the acid used in step (A) is a homogeneous composition comprising a hydrochloride salt of a neutral amino acid, water, and an alcohol, and is a semi-solid composition at 23°C.

8. The detection method according to any one of claims 1 to 7, wherein in step (B), the biological sample mixture is stored for 96 hours or less.

9. The detection method according to any one of claims 1 to 8, wherein in step (B), the biological sample mixture is stored at a temperature of 1 to 30°C.

10. 5. The detection method according to claim 3 or 4, wherein the base used in step (C) is trishydroxymethylaminomethane, N-[tris(hydroxymethyl)methyl]glycine, triethanolamine, sodium hydroxide, potassium hydroxide, calcium hydroxide, N,N-bis(2-hydroxyethyl)glycine, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid, or 2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid.

11. The method according to any one of claims 1 to 10, wherein in step (E), the method for detecting microRNA is a microarray method.

12. The detection method according to any one of claims 1 to 11, wherein the microRNA is a microRNA used as a disease marker.

13. The detection method according to claim 12, wherein the disease is cancer or dementia.

14. 2. A homogeneous composition comprising a hydrochloride salt of a neutral amino acid, water, and an alcohol, which is used as the acid in step (A) of the detection method according to claim 1, and which is semi-solid at 23°C.

15. The composition according to claim 14, wherein the weight ratio of the neutral amino acid hydrochloride in the composition is 65 wt% or more and 70 wt% or less, and the weight ratio of the alcohol in the composition is 2 wt% or more and 6 wt% or less.

16. 16. The composition of claim 14 or 15, wherein the neutral amino acid hydrochloride is glycine hydrochloride or alanine hydrochloride.

17. The composition according to any one of claims 14 to 16, wherein the alcohol is methanol or ethanol.

18. A container for storing a biological sample, in which the composition according to any one of claims 14 to 17 is enclosed.