CN117043353A - Method for detecting microRNA in biological sample, composition of hydrochloride of neutral amino acid and container - Google Patents
Method for detecting microRNA in biological sample, composition of hydrochloride of neutral amino acid and container Download PDFInfo
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- CN117043353A CN117043353A CN202280022502.7A CN202280022502A CN117043353A CN 117043353 A CN117043353 A CN 117043353A CN 202280022502 A CN202280022502 A CN 202280022502A CN 117043353 A CN117043353 A CN 117043353A
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Abstract
If the step of storing is performed after the collection of the biological sample containing the microRNA, the detection amount is reduced. In addition, it takes a relatively long time to dissolve the hydrochloride salt of neutral amino acid as crystals in water. The hydrochloride salt of a neutral amino acid is an acidic substance, has skin irritation and mucous membrane irritation, and thus requires attention in terms of safety in handling. The present invention is a method for detecting microRNA in a biological sample, comprising the steps of: a step of mixing an acid with a biological sample, a step of storing for a predetermined period of time, a step of collecting micrornas in the biological sample, and a step of detecting. Furthermore, the present invention provides a homogeneous semi-solid like composition comprising the hydrochloride salt of a neutral amino acid, water and an alcohol.
Description
Technical Field
The present invention relates to a method for detecting micrornas in a biological sample. Furthermore, the present invention relates to a composition comprising the hydrochloride salt of a neutral amino acid.
Background
Recent advances in gene analysis technology have led to attempts to detect micrornas in biological samples, and to use them in diagnosis, treatment, and the like of diseases. By detecting micrornas associated with diseases from biological samples such as blood, there is a possibility that diseases can be detected more early.
On the other hand, micrornas contained in biological samples such as blood are very small compared to other RNAs, and are easily decomposed by RNA-decomposing enzymes contained in biological samples, as compared to general RNAs which have been studied variously so far. In particular, micrornas that are markers in cancer diagnosis are particularly susceptible to RNA-degrading enzymes when compared with general intracellular RNAs because they exist outside cells in serum or body fluid.
Therefore, a biological sample used for the detection of micrornas is usually used for the detection immediately after the biological sample is obtained. Alternatively, after a biological sample is obtained, the biological sample is stored in a state in which the degradation of micrornas is suppressed by a method such as freezing, and then used for detection. Thus, in the detection of micrornas, handling of biological samples is more important than in the case of other RNAs.
In many cases, a clinical site for acquiring a biological sample in an industrial site is separated from an inspection mechanism for detecting micrornas, and it is necessary to store the biological sample for a long period of time, such as several hours to several days, from the clinical site to the inspection mechanism.
The biological sample used for the detection of microRNA is generally stored by freezing at an ultralow temperature of-70℃or lower. However, a freezing apparatus and a transport material for maintaining a biological sample at an ultralow temperature are high-priced, and a method for preserving a biological sample used for detection of micrornas for a long period of time by a method other than freezing is required.
As a method for inhibiting the degradation of RNA in a biological sample, a method of adding guanidine salt to a biological sample to denature RNA-degrading enzymes and a method of mixing and synthesizing a small molecule inhibitor are known. However, as a more effective and simple method, a method of adjusting the pH of a biological sample to a pH region where the activity of RNA-degrading enzyme is reduced and suppressing degradation of RNA is known.
Patent document 1 describes a method of using RNA by adjusting the pH of a biological sample to 4.0 or less, which is obtained by reducing the activity of RNA-degrading enzyme, thereby inhibiting degradation of RNA, inactivating the RNA-degrading enzyme, and then adjusting the pH of the biological sample to more than 6.0.
The hydrochloride salt of a neutral amino acid comprising glycine hydrochloride is mainly a compound used in the biochemical field. In particular, since the antibody has a property of reversibly dissociating the binding between the antibody and the antigen, the antibody is used for, for example, elution of the antibody during affinity purification of the antibody, washing of the antibody 1 time in multiplex immunostaining, and examination of autoantibodies bound to erythrocytes.
In addition, they are also used in fields other than biochemistry, and have a property of solubilizing substances which are insoluble in water, and therefore are also used for solubilizing poorly soluble polysaccharides (non-patent document 1).
Prior art literature
Patent literature
Patent document 1: japanese patent laid-open No. 8-508637
Non-patent literature
Non-patent document 1: jornal of Applied Polymer Science,123, 3772-3780 (2011).
Disclosure of Invention
Problems to be solved by the invention
In order to diagnose and treat diseases using micrornas, a method for detecting micrornas which are extremely trace and easily decomposed without reducing the detection amount is required. The present invention addresses such a need and provides a method for detecting a biological sample containing micrornas without reducing the amount of micrornas detected even after a long-term storage process.
In addition, glycine hydrochloride is a crystalline solid at normal temperature. In the case of using the aqueous solution for the above-mentioned applications, the aqueous solution is often used. The aqueous glycine hydrochloride solution is prepared by dissolving a solid (crystal) of glycine hydrochloride in water (distilled water or the like), and requires a relatively long time for complete dissolution, and therefore requires sufficient stirring. Further, glycine hydrochloride is an acidic substance and has skin irritation and mucous membrane irritation, and therefore, it is necessary to prevent scattering in a solid state (powdery state), splash in an aqueous solution state, and the like, and particularly, in the handling in the case of movement and the like, attention is required in terms of safety. The present invention provides a composition that can be used simply and safely in the method for detecting micrornas.
Means for solving the problems
The present inventors have first compared the amounts of micrornas detected in a biological sample stored for a long period of time without freezing before and after storage in order to verify whether the micrornas can be detected from the biological sample stored for a long period of time by a method other than freezing before and after storage.
In the case of diagnosing and treating a disease, the following operations are performed: the quantitative value of the specific microRNA or the value calculated by the combination of the quantitative values is used for diagnosis and treatment. In this case, for example, if the detected amount of each microrna is reduced to less than 70%, significant impairment may occur in the judgment and treatment of the disease, and even if the detected amount is reduced to less than 90%, the impairment may occur. Here, since the combinations of micrornas to be quantified are different depending on the disease or treatment of the subject, it is not realistic to find a condition that the fluorescence intensity is not reduced for all micrornas. Accordingly, a method is known in which, instead of measuring a change in the detected amount for each microrna, a change in the detected amount of all micrornas disposed in a microarray is estimated by using the sum of fluorescence intensities of all micrornas detected by the microarray. The inventors of the present invention studied using the sum of fluorescence intensities of all micrornas on a microarray as a detection amount of micrornas, and set a detection amount of 70% before storage of a biological sample as a threshold value that can be used for disease judgment and treatment.
As in comparative example 4 described below, after serum was conditioned and stored at 23℃for 48 hours, the microRNA was detected, and as a result, the detected amount of microRNA was reduced to 28.9% before storage (comparative example 5). That is, it was revealed that in a biological sample stored for a long period of time without freezing, the amount of microRNA detected was significantly reduced as a result of the degradation by the RNA-degrading enzyme compared with that before the storage.
As a result of intensive studies to overcome the above problems, the present inventors have found that micrornas equivalent to or more than those before storage can be detected by mixing an acid with a biological sample, storing the mixture for a predetermined period of time, and then recovering the micrornas.
Further, the inventors have conducted intensive studies to find a composition of a hydrochloride of a neutral amino acid having higher solubility in order to solve the problems of dissolution rate and safety in glycine hydrochloride, and as a result, have found a homogeneous semi-solid composition composed of a hydrochloride of a neutral amino acid, water and an alcohol, and have found that the composition has improved water solubility compared with the conventional solid (crystalline) form of a hydrochloride of a neutral amino acid.
That is, the present invention provides the following (1) to (18).
(1) A method for detecting microRNA in a biological sample, comprising the steps of:
(A) A step of mixing an acid with a biological sample to obtain a biological sample mixed solution;
(B) A step of storing the biological sample mixture obtained in the step (A);
(D) A step of recovering microRNA from the biological sample mixture obtained in the step (B); and
(E) Detecting the microRNA recovered in the step (D).
(2) According to the detection method of (1), in the 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), comprising the following step (C) before the step (D): the biological sample mixture obtained in the step (B) is mixed with a base, and the pH is adjusted to 6.0 to 9.0.
(4) According to the detection method of (3), in the step (C), the pH is adjusted to 7.5 to 9.0.
(5) The detection 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 in the step (A), the acid used 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 in the step (A), the acid used is a homogeneous composition comprising a hydrochloride salt of a neutral amino acid, water and an alcohol, and is a semisolid composition at 23 ℃.
(8) The detection method according to any one of (1) to (7), wherein in the step (B), the biological sample mixture is stored for a period of time of 96 hours or less.
(9) The detection method according to any one of (1) to (8), wherein in the step (B), the temperature of the biological sample mixture is kept at 1 to 30 ℃.
(10) The detection method according to (3) or (4), wherein in the step (C), the base used is tris (hydroxymethyl) aminomethane, N- [ tris (hydroxymethyl) methyl ] glycine, triethanolamine, sodium hydroxide, potassium hydroxide, calcium hydroxide, N-bis (2-hydroxyethyl) glycine, N- (2-hydroxyethyl) piperazine-N' -2-ethane sulfonic acid or 2-hydroxy-3- [ tris (hydroxymethyl) methylamino ] -1-propane sulfonic acid.
(11) The detection method according to any one of (1) to (10), wherein in the 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 method according to (12), wherein the disease is cancer or dementia.
(14) A composition which is a homogeneous composition comprising a hydrochloride salt of a neutral amino acid, water and an alcohol, which is used as an acid in the step (A) of the detection method described in (1), and which is semi-solid at 23 ℃.
(15) The composition according to (14), wherein the weight ratio of the hydrochloride salt of the neutral amino acid in the composition is 65wt% or more and 70wt% or less, and the weight ratio of the alcohol is 2wt% or more and 6wt% 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.
ADVANTAGEOUS EFFECTS OF INVENTION
According to the present invention, after a biological sample containing micrornas is collected, micrornas can be detected without reducing the detection amount even after the biological sample is stored for a long period of time without being frozen, for example.
In addition, the composition of the present invention can be dissolved in water more rapidly than the solid of the hydrochloride salt of a neutral amino acid. Further, since the composition is a homogeneous semisolid composition, there is no safety concern caused by scattering in a solid state (powdery) or splashing of an aqueous solution, which is a problem in the case of a solid or aqueous solution of a hydrochloride of a neutral amino acid, and particularly, handling such as movement is easy.
Detailed Description
The present invention is a method for detecting microRNA in a biological sample, comprising the following steps.
(A) A step of mixing an acid with a biological sample to obtain a biological sample mixture,
(B) A step of storing the biological sample mixture obtained in the step (A) for a predetermined period of time,
(D) A step of recovering microRNA from the biological sample mixture obtained in the step (B),
(E) And (D) detecting the micrornas recovered in the step (D).
Each of the above steps of the present invention will be described below.
The step (a) of the present invention is a step of mixing an acid to obtain a biological sample mixture. The pH of the biological sample is preferably adjusted to 2.0 to 4.0, more preferably to 2.5 to 4.0, whereby the activity of the RNA-degrading enzyme in the biological sample is reduced and the degradation of the microRNA in the biological sample is suppressed. When the pH of the biological sample is adjusted to 2.0 or more, it is preferable to prevent the phosphodiester bond of the microRNA from being cleaved by acid hydrolysis. Further, it is preferable to adjust the pH of the biological sample to 4.0 or less because the enzymatic activity of the RNA-degrading enzyme contained in the biological sample can be sufficiently reduced.
The biological sample used in the present invention refers to a sample obtained from a biological body containing micrornas. Specifically, examples thereof include serum, plasma, whole blood, bone marrow fluid, lymph fluid, saliva, bile, pancreatic juice, ascites fluid, body fluids, secretions, and the like. Cultures such as tissues and cultured cells obtained from other organisms are also possible.
The biological sample is not particularly limited as long as it is collected from a living organism, but is preferably a human-derived substance. The biological sample used in the present invention is particularly preferably human-derived blood, serum or plasma, and more preferably human-derived serum or plasma.
Methods for collecting biological samples are well known in the art. For example, a blood collector such as a medical professional draws blood with a lancet, and then centrifugally separates the blood to obtain serum and plasma.
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 the acid used in the present invention include glycine hydrochloride, alanine hydrochloride, citric acid, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, oxalic acid, and the like. Glycine hydrochloride or citric acid is particularly preferred. In addition, a homogeneous composition comprising a hydrochloride salt of a neutral amino acid, water, an alcohol, and a composition which is semi-solid at 23 ℃ is also preferred.
The method for mixing the biological sample with the acid is not particularly limited. For example, a biological sample may be put into a container in which an acid is previously added, and pipetting and mixing may be performed using a micropipette or the like. Alternatively, for example, a biological sample may be put into a sealable container to which an acid is added in advance, the container may be subjected to a shaking machine such as a vortex to mix the biological sample after the container is sealed, or the container may be turned upside down to mix the biological sample. In particular, the mixing method in which the container is turned upside down can be used to quickly mix the container even in an environment where special devices such as a physical examination vehicle used for regular health examination are not used.
The step (B) of the present invention is a step of storing the biological sample mixture for a predetermined period of time. The predetermined time in this step is a time that can exert the effect of suppressing the decomposition of the micrornas in the biological sample due to the mixing of the acids, and can be appropriately determined in consideration of the time until the step (D).
For example, in the case where the operation is temporarily stopped after the step (a), the biological sample mixture obtained in the step (a) is stored, and then the collection and detection operation of the micrornas is performed, the stored time may be set to a predetermined time of the step. For example, when the biological sample mixture obtained in the step (a) is temporarily stored and then transported, the total time of the storage time and the transport time may be set to a predetermined time in the step.
More specifically, for example, in the case where the biological sample is collected by the hemostix and the place where the microrna is collected and detected is separated as in the case of a regular physical examination, and the biological sample mixture is stored and transported in a place where the biological sample mixture is collected in a sealable container, the storage time and the transport time may be set to the predetermined time in the step.
Specifically, the predetermined time is preferably 1 hour or more, and in particular, in the case of actual use, it is more preferably 24 hours or more, and even more preferably 48 hours or more, if the place where the biological sample is obtained by the hemostix and the place where the collection and detection operation of the micrornas are performed are considered to be separated.
On the other hand, the time for obtaining the effect of the present invention can vary depending on the temperature and the type of biological sample, but is preferably within about 96 hours as the predetermined time.
The preservation of the biological sample mixture in step B may be performed without requiring temperature conditions, such as freezing conditions, under which the biological sample is usually collected and transported. Specifically, the temperature is 1 to 30℃and preferably 15 to 25 ℃. In particular, it is preferable that the storage conditions in the detection of the micrornas in the step (E) are such that a detection amount of 70% or more of the micrornas can be obtained in the microarray method. Further preferably, the storage condition is one under which a detected amount of 90% or more of the microRNA can be obtained.
In this step, the step of adding a base to the biological sample mixture to adjust the pH is preferably performed as the step (C) before the step (D). The pH adjusted in the step (C) is preferably 6.0 to 9.0, which enables detection of the same microRNA as before the storage. The pH is preferably 7.5 to 9.0, more preferably 7.5 to 8.5, which enables detection of microRNAs equal to or more than that before storage. When the pH of the biological sample mixture is adjusted to be less than 6.0 or more than 9.0, the amount of micrornas detected may be reduced as compared with before storage.
The base mixed in the step (C) is a base which can be mixed with the biological sample mixed solution to have a pH of 6.0 to 9.0. Examples thereof include Tris (hydroxymethyl) aminomethane (Tris), N- [ Tris (hydroxymethyl) methyl ] glycine (maifane), triethanolamine, sodium hydroxide, potassium hydroxide, calcium hydroxide, N-bis (2-hydroxyethyl) glycine (Bicine), 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid (HEPES), 2-hydroxy-3- [ Tris (hydroxymethyl) methylamino ] -1-propane sulfonic acid (TAPSO), and the like. As the base used in the present invention, tris (hydroxymethyl) aminomethane is particularly preferred.
The method for mixing the biological sample mixture with the alkali is not particularly limited. For example, the biological sample mixture may be pipetted and mixed by adding an alkali to a container in which the biological sample mixture is previously added, using a micropipette or the like. Alternatively, for example, the biological sample mixture may be mixed by charging alkali into a sealable container in which the biological sample mixture is previously charged and sealing the container, and then supplying the container to a shaker such as a vortex, or by turning the container upside down.
The step (D) of the present invention is a step of extracting and recovering microRNA. The recovery method used may be applied to methods well known in the technical field related to nucleic acid recovery. Examples thereof include phenol/chloroform extraction, column chromatography, ethanol precipitation, and magnetic bead adsorption.
The step (E) of the present invention is a step of detecting the recovered microRNA. The detection method used may be any method known in the art relating to nucleic acid detection. Examples thereof include microarray methods, PCR methods, northern blotting methods, and sequence analysis methods. As the method used in the present invention, a microarray method is particularly preferred.
Examples of the micrornas of the present invention include micrornas used as disease markers. The present invention can provide information for diagnosing a disease by providing a detection value of the expression level in a sample of micrornas that are markers of the disease. Examples of the disease marker of the present invention include markers for cancer and dementia.
Furthermore, the present invention relates to a composition comprising a hydrochloride salt of a neutral amino acid, water, and an alcohol.
The composition of the present invention is characterized by being homogeneous and semi-solid at 23 ℃, unlike a solid (crystal) of a hydrochloride salt of a neutral amino acid, an aqueous solution of a hydrochloride salt of a neutral amino acid, and a suspension in which a solid of a hydrochloride salt of a neutral amino acid is dispersed.
As the acid used in the step (A) of the present invention, a homogeneous composition comprising a hydrochloride of a neutral amino acid, water and an alcohol and a composition which is semi-solid at 23℃is preferable because it can be dissolved in a biological sample mixture rapidly and is not easily scattered
The semisolid is a state in which the deformation is easy by applying a force, and the deformed shape is maintained even after the force is removed. The hydrochloride (crystal) of a neutral amino acid is solid and deformation by force is not easy, and therefore does not correspond to the present definition. In addition, the suspension of the hydrochloride salt of the neutral amino acid does not maintain the deformed shape, and therefore does not correspond to the present definition.
The judgment of whether or not the composition is semi-solid can be made by whether or not the composition retains its deformed shape after being deformed by applying a force. Specifically, the composition was deformed into a cube shape having a height of 1.0cm and a side of 0.5cm, and then, was judged to be semi-solid when the height was maintained at 23℃for 10 minutes or more at 0.8 cm.
The composition of the present invention is characterized by being dissolved in water rapidly at normal temperature without stirring or the like, compared with the solid of the hydrochloride of a neutral amino acid.
The evaluation of whether or not the composition is rapidly dissolved in water can be performed by observing the case where the composition is dissolved by adding to water. Distilled water 10 times the weight of the composition was added to the container in which the composition was sealed, and after standing for a certain period of time, if no solid residue was observed in the water, it was judged to be rapidly dissolved, and if solid residue was observed in the water, it was judged to be undissolved. Specifically, 1mL of distilled water was added to a 2mL PP tube filled with 0.1g of the composition, and after standing at 23 ℃ for 1 minute, the PP tube was judged to be rapidly dissolved if no solid residue was observed in the aqueous solution, and was judged to be undissolved if solid residue was observed in the aqueous solution.
When the glycine hydrochloride solid (crystal) was evaluated by the above method, the solid remained in the aqueous solution, and therefore, it was judged as undissolved (see comparative example 11).
The composition of the invention is a composition which comprises hydrochloride of neutral amino acid, water and alcohol and is semisolid at 23 ℃.
The composition of the present invention preferably comprises a hydrochloride salt of a neutral amino acid in an amount of 65wt% to 70wt%, an alcohol in an amount of 2wt% to 6wt%, and water. The weight ratio of the hydrochloride of the neutral amino acid to the alcohol in the composition can be calculated by diluting the composition with water and then analyzing the diluted composition 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 water vaporizing device and measuring the amount of vaporized water by a karl fischer moisture meter.
The hydrochloride of the neutral amino acid used in the composition of the present invention contains the hydrochloride of an amino acid having the same number of basic amino groups and acidic carboxyl groups contained in the molecule. For example, the amino acids constituting the protein include, but are not limited to, hydrochlorides of glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, asparagine, glutamine, proline, phenylalanine, tyrosine, tryptophan, and the like. Glycine hydrochloride or alanine hydrochloride is preferred from the standpoint of cost and availability.
The alcohol used in the composition of the present invention is preferably an alcohol having 1 to 8 carbon atoms, for example. The alcohol of the present invention is preferably 1 to 3-membered alcohol. Specifically, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, t-butanol, 3-methyl-1-butanol, 1-pentanol, 2-pentanol, n-hexanol, cyclohexanol, 1-heptanol, 1-octanol, 2-methoxyethanol, 1-alcohols such as allyl alcohol, furfuryl alcohol, phenol, and the like, polyols such as ethylene glycol, propylene glycol, butylene glycol, and glycerin, and the like are mentioned, but methanol or ethanol is preferable from the viewpoint of cost and availability.
In the case of a composition (aqueous solution) composed of only glycine hydrochloride and water, the composition having a weight ratio of glycine hydrochloride of more than 50% by weight is insoluble in water at 23 ℃ and becomes a suspension in which a solid of glycine hydrochloride is dispersed in water. In the same manner, in the case of a composition (aqueous solution) composed of only alanine hydrochloride and water, the alanine hydrochloride is insoluble in water at 23 ℃ in a composition having a weight ratio of alanine hydrochloride of more than 50wt%, and the alanine hydrochloride is a suspension in which a solid is dispersed in water. The composition of the present invention contains an alcohol in addition to the hydrochloride salt of a neutral amino acid and water, and as a result, is obtained as a homogeneous semisolid composition, not as a suspension.
The composition of the present invention can be dissolved in water to prepare an aqueous solution of a hydrochloride salt of a neutral amino acid having a desired concentration in a simple manner. In addition, the solvent may be dissolved in an aqueous solvent. Examples of the aqueous solvent include a buffer solution, a medium, and a liquid biological sample. Examples of the liquid biological sample include blood, serum, plasma, urine, saliva, and tears.
Examples of the method for dissolving the composition of the present invention in a solvent include a method for adding the composition to a solvent and a method for adding a solvent to a container in which the composition is sealed in advance. The container in which the composition is sealed in advance is not particularly limited, and examples thereof include plastic pipes, glass pipes, and the like.
The container in which the composition of the present invention is sealed in advance in this way may be a container for storing a biological sample. Such a container has an advantage that the composition is not easily scattered even when unsealed, and stabilization by acidification can be rapidly performed when a biological sample is added.
The amount of the composition of the present invention dissolved in the solvent may be appropriately adjusted depending on the target concentration, but is preferably 0.01g to 10.0g, more preferably 0.1g to 1.0g, per 100mL of the solvent.
Since the 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 the hydrochloride of the conventional neutral amino acid.
In the case of a composition containing glycine hydrochloride, water and methanol or ethanol, for example, the composition of the present invention can be prepared by the following steps.
Step (1): mixing glycine hydrochloride, water and methanol or ethanol in a predetermined weight ratio to obtain a glycine hydrochloride suspension;
Step (2): a step of heating the suspension obtained in (1) to dissolve glycine hydrochloride;
step (3): a step of cooling the glycine hydrochloride solution obtained in (2) to obtain a composition;
the glycine hydrochloride to be mixed in the step (1) is 65 to 70wt% and the ethanol or methanol is 2 to 6 wt%.
In the case of the preparation by the above method, glycine hydrochloride is not dissolved in the suspension of glycine hydrochloride in water in the step (1), but the glycine hydrochloride is dissolved in a homogeneous solution by heating in the step (2), and the solution is cooled in the step (3), whereby a targeted homogeneous semisolid composition is obtained.
In the step (1), the method of mixing glycine hydrochloride, water and ethanol or methanol is not particularly limited. For example, a method of mixing for 1 minute at 23℃using a vortex mixer is mentioned.
In the step (2), the temperature at which the suspension is heated may be a temperature at which the substance having the lowest boiling point in the components is not higher than the boiling point and glycine hydrochloride can be dissolved. For example, ethanol (boiling point 78.4 ℃) may be used, and methanol (boiling point 64.7 ℃) may be used, and heating may be performed at 75℃and 60 ℃. The method of heating is not particularly limited, but a water bath or a block incubator may be used. The heating time is preferably 5 minutes to 30 minutes. In the heating, it is preferable to heat in a sealed state in order to prevent evaporation of water and ethanol or methanol.
In the step (2), the determination of whether glycine hydrochloride is dissolved or not can be performed by visual observation. After heating, the dissolution was judged when no solid residue was observed in the aqueous solution, and the non-dissolution was judged when the solid residue was observed in the aqueous solution.
In the step (3), a general-purpose cooling device may be used as the method for cooling, but the heated aqueous solution may be allowed to stand still directly in a heated container and cooled, or may be transferred to another container and allowed to stand still and cooled. The cooling time is preferably 10 minutes to 60 minutes. The temperature after cooling is preferably 20 to 25 ℃.
The composition of the present invention can also be prepared from alanine hydrochloride by the same procedure as glycine hydrochloride.
The composition of the present invention can be prepared by the same procedure as for the hydrochloride of a neutral amino acid other than glycine hydrochloride and alanine hydrochloride.
Examples
Example 1 detection of micrornas from serum preserved after mixing glycine hydrochloride 1
(A) Mixing an acid with a biological sample
Blood is collected from the healthy subject X, and serum is prepared. To 300. Mu.L of the immediately prepared serum, 90. Mu.L of 1M glycine hydrochloride aqueous solution (Fujifei co and Fujiaye, inc.) was mixed. The pH of the serum was measured using a portable pH meter (horiba, ltd.) and, as a result, it was confirmed that the pH was lowered from 7.5 to 2.8 by mixing the acid.
(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℃for 48 hours.
(C) Mixing alkali in biological sample mixture
To the serum stored in the step (B), 27 μl of 4M aqueous solution of tris (hydroxymethyl) aminomethane (nux corporation) was mixed. Using a portable pH meter, it was confirmed that the pH increased from 2.8 to 7.5 by mixing of the base.
(D) Process for recovering microRNA from biological sample mixture
From the serum obtained by mixing the base in the step (C), microRNA was collected by using a reagent for RNA extraction of 3D-Gene (registered trademark) RNA extraction reagent from liquid sample kit (Togaku Co., ltd.) according to an experimental method prescribed by the company.
(E) Detecting microRNA
The micrornas recovered from serum in step (D) were fluorescently labeled using 3D-Gene (registered trademark) miRNA Labeling kit (by kogaku corporation) based on an experimental method prescribed by the company. As a DNA microarray chip, 3D-Gene (registered trademark) Human miRNA Oligo chip (da corporation) on which a probe having a sequence complementary to 2,565 micrornas was mounted was used, and hybridization was performed based on an experimental method prescribed by the company. The DNA microarray chip was scanned with a 3D-Gene (registered trademark) scanner (produced by Toyo-ro Co., ltd.) to obtain an image. The fluorescence intensity was quantified using 3D-Gene (registered trademark) Extraction, and the total value of the fluorescence intensities of micrornas detected exceeding the detection limit was calculated and used as the detection amount of micrornas. The detected amounts of micrornas are shown in table 1.
EXAMPLES 2 to 5 detection of microRNA from serum preserved after mixing glycine hydrochloride 2
In the course of the mixing of the bases,
in example 2 the pH was adjusted to 7.7 by mixing 30 μl of 4M aqueous tris (hydroxymethyl) aminomethane solution,
in example 3 the pH was adjusted to 8.0 by mixing 36 μl of 4M aqueous tris (hydroxymethyl) aminomethane solution,
in example 4 the pH was adjusted to 8.5 by mixing 60 μl of 4M aqueous tris (hydroxymethyl) aminomethane solution,
in example 5 the pH was adjusted to 9.0 by mixing 120 μl of 4M aqueous tris (hydroxymethyl) aminomethane solution,
except for this, the same procedure as in example 1 was performed. The detected amounts of the micrornas of examples 2 to 5 are shown in table 1.
Examples 6 and 7 detection of microRNA from serum preserved after mixing glycine hydrochloride 3
In the course of the mixing of the bases,
in example 6 the pH was adjusted to 6.0 by mixing 21 μl of 4M aqueous tris (hydroxymethyl) aminomethane solution,
in example 7 the pH was adjusted to 7.0 by mixing 22.5 μl of 4M aqueous tris (hydroxymethyl) aminomethane,
except for this, the same procedure as in example 1 was performed. The detected amounts of micrornas of examples 6 and 7 are shown in table 1.
Example 8 detection of microRNA from serum stored after mixing glycine hydrochloride without mixing alkali
In example 8, the same procedure as in example 1 was carried out except that the alkali was not mixed. The detected amounts of the micrornas of example 8 are shown in table 1.
Comparative example 1 detection of microRNA from serum stored without mixing acid and without mixing alkali
In comparative example 1, the same procedure as in example 1 was carried out except that the acid and the base were not mixed. The detected amounts of micrornas of comparative example 1 are shown in tables 1 and 2.
Comparative example 2 detection of microRNA from serum immediately after preparation 1
From 300. Mu.L of serum collected from a healthy subject X, microRNAs were collected in the same manner as in the step (D) of example 1 without performing the steps (A) to (C) of example 1, and the microRNAs collected in the same manner as in the step (E) of example 1 were detected. The detected amounts of micrornas are shown in tables 1 and 2.
TABLE 1
TABLE 1 detection amount of microRNAs of examples 1 to 8 and comparative examples 1 and 2
The micrornas were detected to be equal to or higher than (100% or higher) the case where the ph was adjusted to 2.8 by mixing the acid with serum, then stored at 23 ℃ for 48 hours, and then adjusted to ph7.5 to 8.5 by mixing the alkali (examples 1 to 4) compared with the case where the serum immediately after the preparation was used (comparative example 2). Even when the pH was adjusted to 9.0 by mixing the alkali (example 5), 90% or more of microRNAs were detected as compared with the case where the serum immediately after the preparation was used (comparative example 2). In addition, even when pH was adjusted to 6.0 and 7.0 by mixing alkali (examples 6 and 7), microRNAs equivalent (70% or more) were detected as compared with the case where serum immediately after the preparation was used (comparative example 2). Further, even in the case where no alkali was mixed (example 8), micrornas equivalent (70% or more) were detected as compared with the case where serum immediately after preparation was used (comparative example 2).
On the other hand, when the sample was stored at 23℃for 48 hours without mixing the acid with serum (comparative example 1), the amount of microRNA detected was reduced to less than 30% as compared with the case where the serum immediately after the preparation was used (comparative example 2). This is considered to be the result of the decomposition of micrornas during storage, since no acid is mixed.
From the above results, it is clear that preservation of serum after mixing with an acid is important for detection of micrornas, and in particular, adjustment of pH to 7.5 to 9.0 after mixing with an alkali after preservation is important for detection of micrornas.
Example 9 detection of micrornas from serum preserved after mixing glycine hydrochloride 4
The same procedure as in example 1 was conducted except that 45. Mu.L of 1M glycine hydrochloride aqueous solution was mixed to adjust the pH to 4.0 at the time of acid mixing, and 10. Mu.L of 4M aqueous solution of tris (hydroxymethyl) aminomethane was mixed to adjust the pH to 7.7 at the time of alkali mixing. The detected amounts of micrornas are shown in table 2.
Comparative example 3 detection of micrornas from serum preserved without mixing acid
In comparative example 3, the same procedure as in example 1 was conducted except that 0.75. Mu.L of a 4M aqueous solution of tris (hydroxymethyl) aminomethane was mixed without mixing the acid and the pH was adjusted to 7.7 at the time of alkali mixing. The detected amounts of micrornas are shown in table 2.
TABLE 2
TABLE 2 detection amount of microRNAs of example 9 and comparative examples 1 to 3
The micrornas were detected to be equal to or higher than (90% or higher) the case where the ph was adjusted to 4.0 by mixing the acid with serum, then stored at 23 ℃ for 48 hours, and then adjusted to ph7.7 by mixing the alkali (example 9) compared with the case where the serum immediately after the preparation was used (comparative example 2).
On the other hand, when the serum was stored at 23℃for 48 hours without mixing the acid, the pH was adjusted to 7.7 by mixing the alkali (comparative example 3) and when the serum was not mixed with the alkali (comparative example 1), the amount of microRNA detected was reduced to less than 30% as compared with the case where the serum immediately after the preparation was used (comparative example 2). This is considered to be the result of the decomposition of micrornas during storage, since no acid is mixed.
From the above results, it was found that mixing an acid in serum before storage and adjusting pH to 4.0 or less is important for detection of microRNA.
Examples 10 to 12 detection of microRNA from serum preserved after mixing glycine hydrochloride 5
The preservation time of the biological sample mixture is prolonged,
in example 10 for 2 hours,
in example 11 for 6 hours, and
in example 12 for 96 hours,
in examples 10 to 12, the same procedure as in example 1 was carried out except that 30. Mu.L of a 4M aqueous solution of tris (hydroxymethyl) aminomethane was mixed at the time of alkali mixing to adjust the pH to 7.7. The detected amounts of the microRNAs in examples 10 to 12 are shown in Table 3.
TABLE 3
TABLE 3 detection amounts of microRNAs of examples 10 to 12 and comparative example 2
The samples were mixed with acid to adjust the pH to 2.8, stored at 23℃for 2, 6 and 96 hours, and then mixed with alkali to adjust the pH to 7.7 (examples 10 to 12), and microRNAs equivalent to or higher (100% or higher) were detected as compared with the samples obtained by using the freshly prepared serum (comparative example 2).
From the above, it is clear that microRNA detection can be performed from serum stored at 23℃for at least 2 to 96 hours by using the present method.
Example 13 detection of micrornas from serum preserved after mixing citric acid 1
The same procedure as in example 1 was carried out except that the serum collected from the healthy subject Y was used, 90 μl of 1M aqueous citric acid (Sigma-Aldrich co.llc.) was mixed at the time of acid mixing to adjust the pH to 3.1, and 90 μl of 4M aqueous tris (hydroxymethyl) aminomethane was mixed at the time of alkali mixing to adjust the pH to pH 7.8. The detected amounts of micrornas are shown in table 4.
Examples 14 and 15 detection of micrornas from serum preserved after mixing citric acid 2
In the course of the mixing of the bases,
in example 14, 63. Mu.L of a 4M aqueous solution of tris (hydroxymethyl) aminomethane was mixed and the pH was adjusted to 6.0,
In example 15, 67.5. Mu.L of a 4M aqueous solution of tris (hydroxymethyl) aminomethane was mixed and adjusted to pH7.0,
except for this, the same procedure as in example 13 was carried out. The detected amounts of micrornas are shown in table 4.
EXAMPLE 16 detection of microRNA from serum preserved after mixing citric acid without mixing alkali
The same procedure as in example 13 was carried out except that the alkali was not mixed. The detected amounts of micrornas are shown in table 4.
Comparative example 4 detection of micrornas from freshly prepared serum 2
From 300. Mu.L of serum collected from a healthy subject Y, microRNAs were collected in the same manner as in the step (D) of example 1 without performing the steps (A) to (C) of example 1, and the microRNAs collected in the same manner as in the step (E) of example 1 were detected. The detected amounts of micrornas are shown in table 4.
TABLE 4
TABLE 4 detection amount of microRNAs of examples 13 to 16 and comparative example 4
When citric acid was mixed with serum, the mixture was stored at 23℃for 48 hours, and then, alkali was mixed and the pH was adjusted to pH7.8 (example 13), microRNAs equivalent to or higher than that obtained when freshly prepared serum was used (comparative example 4) (100% or higher) were detected. Even when the pH was adjusted to 6.0 or 7.0 by mixing the alkali after storage (examples 14 and 15) and when the alkali was not mixed (example 16), microRNAs equivalent (70% or more) to those obtained by using the serum immediately after the preparation (comparative example 4) were detected.
From the above results, it was confirmed that the acid mixed in the serum was independent of the kind, and that glycine hydrochloride and citric acid were effective.
EXAMPLE 17 detection of microRNA from serum preserved after mixing Glycine hydrochloride 6
The same procedure as in example 1 was carried out except that the serum collected from the healthy subject Z was used, 180. Mu.L of 1M glycine hydrochloride aqueous solution was mixed at the time of acid mixing to adjust the pH to 2.0, and 60. Mu.L of 4M tris aqueous solution was mixed at the time of alkali mixing to adjust the pH to 7.7. The detected amounts of micrornas are shown in table 5.
Comparative example 5 detection of micrornas from freshly prepared serum 3
From 300. Mu.L of serum collected from a healthy subject Z, microRNAs were recovered by the same method as in the step (D) of example 1 without performing the steps (A) and (B) of example 1, and the recovered microRNAs were detected in the same manner as in the step (E) of example 1. The detected amounts of micrornas are shown in tables 5 and 6.
TABLE 5
TABLE 5 detection amount of microRNAs of example 17 and comparative example 5
The micrornas were detected to be equal to or higher than (100% or higher) the case where the ph was adjusted to 2.0 by mixing the acid with serum, then stored at 23 ℃ for 48 hours, and then adjusted to ph7.7 by mixing the alkali (example 17) compared with the case where the serum immediately after the preparation was used (comparative example 5).
From the above results, it was found that mixing an acid in serum before storage and adjusting the pH to at least 2.0 to 4.0 is important for detection of microRNAs.
Example 18 detection of micrornas from serum preserved after mixing glycine hydrochloride 7
The same procedure as in example 1 was carried out except that the serum collected from the healthy subject Z was used, the pH was adjusted to 2.8 by mixing the composition 2 described in example 19 described below with an acid, and pH was adjusted to 7.7 by mixing 30 μl of a 4M aqueous solution of tris (hydroxymethyl) aminomethane with a base. The detected amounts of micrornas are shown in table 6.
TABLE 6
TABLE 6 detection amount of microRNAs of example 18 and comparative example 5
When a homogeneous composition 2, which is a semisolid at 23℃and contains a hydrochloride salt of a neutral amino acid, water and an alcohol, was mixed with serum and then stored at 23℃for 48 hours, and then mixed with a base and adjusted to pH7.7 (example 18), microRNAs equal to or higher (100% or higher) than those obtained by using freshly prepared serum (comparative example 4) were detected.
From the above results, it was revealed that the acid mixed in the serum before storage was a homogeneous composition comprising a hydrochloride salt of a neutral amino acid, water and an alcohol, and was a semisolid composition at 23 ℃.
Example 19
Compositions (compositions 1 to 6) comprising glycine hydrochloride, water and ethanol
(1) Preparation of the composition
To a 2mL PP tube, 650mg (65.0 wt%) of glycine hydrochloride (Fusifun and light of Di-Yi Co., ltd.), 20mg (2.0 wt%) of ethanol (Fusifun and light of Di-Yi Co., ltd.) and 330mg (33.0 wt%) of distilled water were added to make the total amount of the mixture 1.0g, and the mixture was mixed at 23℃for 1 minute using a vortex mixer to obtain a suspension. The tube with the suspension added was heated at 75℃for 10 minutes using a block incubator. After heating, the dissolution state of glycine hydrochloride was evaluated by visual observation, and as a result, it was confirmed that no solid residue was observed in the aqueous solution and glycine hydrochloride was dissolved. After the tube was taken out of the block incubator, it was allowed to stand at 23℃for 30 minutes to obtain composition 1.
Compositions 2 to 6 were prepared by the same procedure as described 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.
In the preparation steps of compositions 2 to 6, dissolution of glycine hydrochloride was confirmed by visual observation after heating.
(2) Evaluation of shape
The composition 1 obtained in (1) was deformed into a cube shape with 0.5cm on both sides and 1.0cm in height on a plastic dish using a spatula, and after the deformation, the height was measured after standing at 23℃for 10 minutes, and as a result, the height was 0.8cm or more, and the deformed shape was maintained, so that it was judged that the composition 1 was semisolid.
As a result of evaluating the states of the compositions 2 to 6 by the same method as described above, it was judged that the composition 1 was semi-solid since the deformed shape was maintained in any of the compositions.
(3) Evaluation of solubility
The solubility of the composition 1 obtained in (1) was evaluated. To a 2mL PP tube containing 0.1g of composition 1 was added 1mL of distilled water, and after standing at 23℃for 1 minute, the dissolution state was evaluated by visual observation, and as a result, no solid residue was observed in the aqueous solution, and it was judged that dissolution was rapid.
As a result of evaluating the solubility of the compositions 2 to 6 by the same method as described above, no solid residue was observed in the aqueous solution, and it was therefore judged that the compositions were rapidly dissolved.
Comparative example 6
Compositions (compositions 7 to 20) comprising glycine hydrochloride, water and ethanol
(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.
In the preparation steps of compositions 7 to 15, dissolution of glycine hydrochloride was confirmed by visual observation after heating. On the other hand, in the preparation steps of the compositions 16 to 20, since the remaining of solids was observed in the aqueous solution even after heating, it was judged that a part of glycine hydrochloride was not dissolved, and the subsequent evaluation was not performed.
(2) Evaluation of shape
The state of the compositions 7 to 15 obtained in (1) was evaluated in the same manner as in (2) of example 19, and as a result, it was confirmed by visual observation that the solid was dispersed in the solution, and it was judged that the compositions were not semisolid but suspension, since the deformed shape was not maintained in any of the compositions.
(3) Evaluation of solubility
The compositions 7 to 15 obtained in (1) were evaluated for solubility by the same method as in (3) of example 19, and as a result, precipitation of solids was observed in the aqueous solution, and therefore, it was judged as undissolved.
The results obtained in example 19 and comparative example 6 are shown in table 7.
TABLE 7
TABLE 7 compositions comprising glycine hydrochloride, water and ethanol (compositions 1-20)
Dissolution state of the heated composition: o = dissolved. X = undissolved (residual).
Dissolution state of the composition after cooling: o = dissolved. X=undissolved (precipitated).
Compositions 1 to 6 of example 19 were semi-solid and rapidly dissolved in distilled water. On the other hand, compositions 7 to 15 of comparative example 11 were suspensions in which the precipitated glycine hydrochloride solids were dispersed in an aqueous solution, and were not rapidly dissolved even when distilled water was further added. In addition, compositions 16 to 20 of comparative example 6 did not dissolve glycine hydrochloride even when heated.
From the above results, it was found that when the weight ratio of glycine hydrochloride in the composition was 65wt% or more and 70wt% or less and the weight ratio of ethanol was 2wt% or more and 6wt% or less, a semisolid composition which was rapidly dissolved in water was obtained.
Example 20
Compositions (compositions 21-26) comprising glycine hydrochloride, water and methanol
(1) Preparation of the composition
In example 19 (1), the ethanol was changed to methanol (fuinfrafand light pure) and the heating temperature was changed from 75 ℃ to 60 ℃, and glycine hydrochloride, methanol and distilled water were used in amounts shown in table 8 to prepare compositions 21 to 26.
In the preparation steps of the compositions 21 to 26, dissolution of glycine hydrochloride was confirmed by visual observation after heating.
(2) Evaluation of shape
The state of the compositions 21 to 26 obtained in (1) was evaluated in the same manner as in (2) of example 19, and as a result, the deformed shape of each composition was maintained, and thus, it was judged as semisolid.
(3) Evaluation of solubility
The solubility of the compositions 21 to 26 obtained in (1) was evaluated in the same manner as in (3) of example 19, and as a result, no precipitation of solids was observed in the aqueous solution, and it was determined that the solid was dissolved.
Comparative example 7
Compositions (compositions 27-40) comprising glycine hydrochloride, water and methanol
(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 shown in Table 8.
In the preparation steps of the compositions 27 to 35, dissolution of glycine hydrochloride was confirmed by visual observation after heating. On the other hand, in the preparation steps of the compositions 36 to 40, since the solid remained in the aqueous solution even after heating, it was judged that glycine hydrochloride was not dissolved, and the subsequent operations were not performed.
(2) Evaluation of shape
The state of the compositions 27 to 35 obtained in (1) was evaluated in the same manner as in (2) of example 19, and as a result, it was confirmed by visual observation that the solid was dispersed in the solution, and it was judged that the compositions were not semisolid but suspension, since the deformed shape was not maintained in any of the compositions.
(3) Evaluation of solubility
The compositions 27 to 35 obtained in (1) were evaluated for solubility by the same method as in (3) of example 19, and as a result, precipitation of solids was observed in the aqueous solution, and therefore, it was judged as undissolved.
The results obtained in example 20 and comparative example 7 are shown in table 8.
TABLE 8
TABLE 8 compositions comprising glycine hydrochloride, water and methanol (compositions 21-40)
Dissolution state of the heated composition: o = dissolved. X = undissolved (residual).
Dissolution state of the composition after cooling: o = dissolved. X=undissolved (precipitated).
Compositions 21 to 26 of example 20 were semi-solid and rapidly dissolved in distilled water. On the other hand, compositions 27 to 35 of comparative example 7 were suspensions in which the precipitated glycine hydrochloride was dispersed in an aqueous solution, and were not rapidly dissolved even when distilled water was further added. In addition, compositions 36 to 40 of comparative example 7 did not dissolve glycine hydrochloride even when heated.
As is clear from the above results, in the case of methanol, the glycine hydrochloride was contained in the composition in a weight ratio of 65wt% or more and 70wt% or less, and in the case of methanol in a weight ratio of 2wt% or more and 6wt% or less, the composition was obtained as a semisolid and was rapidly dissolved in water.
Comparative example 8
Preparation and evaluation of compositions (compositions 41 to 52) comprising Glycine hydrochloride, water and acetone
(1) Preparation of the composition
In example 19 (1), compositions 41 to 52 were prepared by changing ethanol to acetone (Fusiffil and light pure), changing the heating temperature from 75℃to 60℃and using glycine hydrochloride, acetone and distilled water in the weights shown in Table 9.
In the preparation steps of compositions 41 to 49, dissolution of glycine hydrochloride was confirmed by visual observation after heating. On the other hand, in the preparation steps of the compositions 50 to 52, since the solid remained in the aqueous solution even after heating, it was judged that glycine hydrochloride was not dissolved, and the subsequent operations were not performed.
(2) Evaluation of shape
The state of the compositions 41 to 49 obtained in (1) was evaluated in the same manner as in (2) of example 19, and as a result, it was confirmed by visual observation that the solid was dispersed in the solution, and it was judged that the compositions were not semisolid but suspension, since the deformed shape was not maintained in any of the compositions.
(3) Evaluation of solubility
The solubility of the compositions 41 to 49 obtained in (1) was evaluated in the same manner as in (3) of example 19, and as a result, precipitation of solids was observed in the aqueous solution, and therefore, it was judged as undissolved.
The results obtained in comparative example 8 are shown in table 9.
TABLE 9
TABLE 9 compositions comprising glycine hydrochloride, water and acetone (compositions 41-52)
Dissolution state of the heated composition: o = dissolved. X = undissolved (residual).
Dissolution state of the composition after cooling: o = dissolved. X=undissolved (precipitated).
When acetone is used instead of ethanol or methanol, the composition obtained is in the form of a suspension of glycine hydrochloride solids dispersed in an aqueous solution, and the composition which is the object of the present invention is not obtained.
Comparative example 9
Compositions (compositions 53-64) comprising glycine hydrochloride, water and acetonitrile
(1) Preparation of the composition
In example 19 (1), ethanol was changed to acetonitrile (fuinfrafand pure light), and compositions 53 to 64 were prepared using glycine hydrochloride, acetonitrile and distilled water in the weights shown in table 10.
In the preparation of compositions 53 to 61, dissolution of glycine hydrochloride was confirmed by visual observation after heating. On the other hand, in the preparation of the compositions 62 to 64, since the solid remained in the aqueous solution even after heating, it was judged that glycine hydrochloride was not dissolved, and the subsequent operations were not performed.
(2) Evaluation of shape
The state of the compositions 53 to 61 obtained in (1) was evaluated in the same manner as in (2) of example 19, and as a result, it was confirmed by visual observation that the solid was dispersed in the solution without maintaining the deformed shape of any of the compositions, and it was judged that the compositions were not semisolid but suspension.
(3) Evaluation of solubility
The solubility of the compositions 53 to 61 obtained in (1) was evaluated in the same manner as in (3) of example 19, and as a result, precipitation of solids was observed in the aqueous solution, and therefore, it was judged as undissolved.
The results obtained in comparative example 9 are shown in table 10.
TABLE 10
Table 10 compositions comprising glycine hydrochloride, water and acetonitrile (compositions 53 to 64)
Dissolution state of the heated composition: o = dissolved. X = undissolved (residual).
Dissolution state of the composition after cooling: o = dissolved. X=undissolved (precipitated).
When acetonitrile is used, the composition obtained is in the form of a suspension of glycine hydrochloride solids dispersed in an aqueous solution, and the composition which is the object of the present invention is not obtained.
Comparative example 10
Compositions (compositions 65 to 68) comprising glycine hydrochloride and water
(1) Preparation of the composition
In example 19 (1), compositions 65 to 68 were prepared by changing the method to a method using no ethanol and using glycine hydrochloride and distilled water in the weight ratio shown in table 11.
During the preparation of the compositions 65 to 67, dissolution of glycine hydrochloride was confirmed by visual observation after heating. On the other hand, since the solid remained in the aqueous solution even after heating during the preparation of the composition 68, it was judged that glycine hydrochloride was not dissolved, and no subsequent operation was performed.
(2) Evaluation of shape
The state of the compositions 65 to 67 obtained in (1) was evaluated in the same manner as in (2) of example 19, and as a result, it was confirmed by visual observation that the solid was dispersed in the solution, and it was judged that the compositions were not semisolid but suspension, since the deformed shape was not maintained in any of the compositions.
(3) Evaluation of solubility
The compositions 65 to 67 obtained in (1) were evaluated for solubility by the same method as in (3) of example 19, and as a result, precipitation of solids was observed in the aqueous solution, and therefore, it was judged as undissolved.
The results obtained in comparative example 10 are shown in table 11.
TABLE 11
TABLE 11 compositions (compositions 65-68) comprising glycine hydrochloride and water
Dissolution state of the heated composition: o = dissolved. X = undissolved (residual).
Dissolution state of the composition after cooling: o = dissolved. X=undissolved (precipitated).
The composition obtained was in the form of a suspension of glycine hydrochloride solids dispersed in an aqueous solution, and the composition targeted by the present invention was not obtained.
As is clear from the results obtained in comparative examples 8 to 10, the semisolid and rapidly water-soluble composition which is the object of the present invention requires ethanol or methanol in addition to glycine hydrochloride and water.
Comparative example 11
Evaluation of solubility of glycine hydrochloride solid
To a 2mL PP tube containing 0.1g glycine hydrochloride was added 1mL of distilled water, and after standing at 23℃for 1 minute, the dissolution state was evaluated by visual observation, and as a result, the remaining of solids was observed in the aqueous solution, and therefore, it was judged as undissolved.
Example 21
pH adjustment of serum Using compositions comprising Glycine hydrochloride, water and ethanol (compositions 1-6)
1mL of serum prepared by taking blood from a healthy person was added to a 2mL tube filled with 0.1g of composition 1 prepared in example 19 (1), and the mixture was left standing at 23℃for 1 minute, and then the dissolved state was evaluated by visual observation, as a result, no solid was observed in the serum, and it was determined that each composition was dissolved rapidly. 0.1mL of the serum was collected from the upper layer of the serum in the tube, and the pH was measured by a portable pH meter (horiba, inc.), and as a result, the pH was 2.7, which was significantly lower than the pH (7.7) of the serum before addition.
By the same method as described above, 1mL of serum was added to a tube in which 0.1g of compositions 2 to 6 was sealed, and the dissolution state was visually evaluated, and as a result, no solid was observed in the serum, it was judged that compositions 2 to 6 were rapidly dissolved. As a result of measurement of pH after standing, the pH was 2.7 for the tubes in which compositions 2 and 3 were sealed, and pH was 2.6 for the tubes in which compositions 4 to 6 were sealed, and was significantly lower than pH 7.7) of serum before addition.
Comparative example 12
pH adjustment of serum using glycine hydrochloride solids
By the same method as in example 21, 1mL of serum was added to a 2mL pp tube filled with 65mg of glycine hydrochloride solid (crystals), and the dissolution state was visually evaluated, and as a result, the solid was observed in the serum, and it was determined that glycine hydrochloride solid was not rapidly dissolved. After standing, the pH of the serum was measured, and as a result, the pH was 4.5, which was reduced to a smaller extent than in the case of example 21 to which compositions 1 to 6 were added.
The results of example 21 and comparative example 12 are shown in table 12.
TABLE 12
TABLE 12 pH adjustment of serum Using compositions comprising Glycine hydrochloride, water and ethanol (compositions 1-6) and Glycine hydrochloride solids
Solubility of the composition: o = dissolved. X = undissolved (residual).
The compositions comprising glycine hydrochloride, water and ethanol (compositions 1 to 6) dissolved rapidly in the serum, lowering the serum pH. On the other hand, glycine hydrochloride solids simply stand, do not dissolve rapidly in serum, and the pH of serum cannot be sufficiently lowered.
From the above, it is clear that the composition of the present invention can be used as a pH adjuster.
Example 22
Compositions comprising alanine hydrochloride, water and ethanol (compositions 69, 70)
In example 19 (1), the glycine hydrochloride was changed to alanine hydrochloride (zebra), and compositions 69 and 70 were prepared using alanine hydrochloride, ethanol, and distilled water in the weights shown in table 13.
In the preparation steps of the compositions 69 and 70, the dissolution of alanine hydrochloride was confirmed by visual observation after heating.
(2) Evaluation of shape
The state of the compositions 69 and 70 obtained in (1) was evaluated in the same manner as in (2) of example 19, and as a result, both compositions remained in the deformed shape, and were thus judged to be semi-solid.
(3) Evaluation of solubility
The solubility of the compositions 69 and 70 obtained in (1) was evaluated in the same manner as in (3) of example 19, and as a result, no precipitation of solids was observed in the aqueous solution, and it was determined that the solids were dissolved.
Comparative example 13
Compositions (compositions 71-80) comprising alanine hydrochloride, water and ethanol
(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.
In the preparation steps of the compositions 71 to 77, the dissolution of alanine hydrochloride was confirmed by visual observation after heating. On the other hand, in the preparation steps of the compositions 78 to 80, since the solids remained in the aqueous solution even after heating, it was judged that alanine hydrochloride was not dissolved, and the subsequent operations were not performed.
(2) Evaluation of shape
The state of the compositions 71 to 77 obtained in (1) was evaluated in the same manner as in (2) of example 19, and as a result, it was confirmed by visual observation that the solid was dispersed in the solution, and it was judged that the compositions were not semisolid but suspension, since the deformed shape was not maintained in any of the compositions.
(3) Evaluation of solubility
The solubility of the compositions 71 to 77 obtained in (1) was evaluated in the same manner as in (3) of example 19, and as a result, precipitation of solids was observed in the aqueous solution, and therefore, it was judged as undissolved.
The results obtained in example 22 and comparative example 13 are shown in table 13.
TABLE 13
TABLE 13 compositions comprising alanine hydrochloride, water and ethanol (compositions 69-80)
Dissolution state of the heated composition: o = dissolved. X = undissolved (residual).
Dissolution state of the composition after cooling: o = dissolved. X=undissolved (precipitated).
Compositions 69 and 70 of example 22, which used alanine hydrochloride, were semi-solid and rapidly dissolved in distilled water. On the other hand, compositions 71 to 77 of comparative example 13 were suspensions in which the solids of the precipitated alanine hydrochloride were dispersed in an aqueous solution, and were not rapidly dissolved even when distilled water was further added. In addition, the alanine hydrochloride was not dissolved in the compositions 78 to 80 of comparative example 13 even when they were heated.
From the above results, it was found that, similarly to the case of glycine hydrochloride, in the case of alanine hydrochloride, the composition was obtained in a semisolid state and rapidly dissolved in water, even when the weight ratio of alanine hydrochloride in the composition was 65wt% or more and 70wt% or less, and the weight ratio of ethanol was 5 wt%.
Comparative example 14
Composition comprising glutamic acid hydrochloride, water and ethanol (compositions 81 to 92)
(1) Preparation of the composition
In example 19 (1), the glycine hydrochloride was changed to glutamic acid hydrochloride, and the compositions 81 to 92 were prepared using glutamic acid hydrochloride, ethanol, and distilled water in the amounts by weight described in table 14.
During the preparation of the compositions 81 to 86, the dissolution of glutamic acid hydrochloride was confirmed by visual observation after heating. On the other hand, in the preparation of the compositions 87 to 92, since the solids remained in the aqueous solution even after heating, it was judged that glutamic acid hydrochloride was not dissolved, and the subsequent operations were not performed.
(2) Evaluation of shape
The state of the compositions 81 to 86 obtained in (1) was evaluated in the same manner as in (2) in example 19, and as a result, neither composition remained in the deformed shape, nor was a solid observed in the solution by visual observation, and therefore it was judged that the composition was not semi-solid but a solution in which glutamic acid hydrochloride was completely dissolved. Since glutamate hydrochloride was dissolved, the evaluation of the solubility as shown in (3) of example 19 was not performed.
The results obtained in comparative example 14 are shown in table 14.
TABLE 14
TABLE 14 compositions comprising glutamic acid hydrochloride, water and ethanol (compositions 81-92)
Dissolution state of the heated composition: o = dissolved. X = undissolved (residual).
Dissolution state of the composition after cooling: o = dissolved. X=undissolved (precipitated).
When glutamic acid hydrochloride is used, the composition obtained is in the form of a solution in which glutamic acid hydrochloride is completely dissolved, and the composition which is the object of the present invention is not obtained.
Comparative example 15
Compositions (compositions 93 to 104) comprising lysine-1 hydrochloride, water and ethanol
(1) Preparation of the composition
In example 19 (1), the glycine hydrochloride was changed to lysine 1 hydrochloride, and compositions 93 to 104 were prepared using lysine 1 hydrochloride, ethanol, and distilled water in the amounts shown in table 15.
In the preparation of the compositions 93 to 98, dissolution of lysine 1 hydrochloride was confirmed by visual observation after heating. On the other hand, since solids remained in the aqueous solution even after heating in the preparation of the compositions 99 to 104, it was judged that lysine 1 hydrochloride was not dissolved, and no subsequent operation was performed.
(2) Evaluation of shape
The state of the compositions 93 to 98 obtained in (1) was evaluated in the same manner as in (2) in example 19, and as a result, neither composition remained in the deformed shape, nor was a solid observed in the solution by visual observation, and it was judged that the composition was not semisolid but a solution in which lysine 1 hydrochloride was completely dissolved. Since lysine 1 hydrochloride was dissolved, the solubility as shown in (3) of example 19 was not evaluated.
The results obtained in comparative example 15 are shown in table 15.
TABLE 15
TABLE 15 composition comprising lysine-1 hydrochloride, water and ethanol
(compositions 93 to 104)
Dissolution state of the heated composition: o = dissolved. X = undissolved (residual).
Dissolution state of the composition after cooling: o = dissolved. X=undissolved (precipitated).
When lysine 1 hydrochloride is used, the composition obtained is in the form of a solution in which lysine 1 hydrochloride is completely dissolved, and the composition which is the object of the present invention is not obtained.
Comparative example 16
Compositions (compositions 105 to 116) comprising lysine 2 hydrochloride, water and ethanol
(1) Preparation of the composition
In example 19 (1), the glycine hydrochloride was changed to lysine 2 hydrochloride (fujifim and light pure), and the compositions 105 to 116 were prepared using the lysine 2 hydrochloride, ethanol and distilled water in the weights shown in table 16.
During the preparation of the compositions 105 to 113, dissolution of lysine 2 hydrochloride was confirmed by visual observation after heating. On the other hand, in the preparation of the compositions 114 to 116, since the solids remained in the aqueous solution even after heating, it was judged that lysine 2 hydrochloride was not dissolved, and the subsequent operations were not performed.
(2) Evaluation of shape
The state of the compositions 105 to 113 obtained in (1) was evaluated in the same manner as in (2) of example 19, and as a result, neither composition remained in the deformed shape, and no solid was observed in the solution by visual observation, so that it was judged that the composition was not semisolid but was a solution in which lysine 2 hydrochloride was completely dissolved. Since lysine 2 hydrochloride was dissolved, the solubility as shown in (3) of example 19 was not evaluated.
The results obtained in comparative example 16 are shown in table 16.
TABLE 16
TABLE 16 composition comprising lysine 2 hydrochloride, water and ethanol
(compositions 105 to 116)
Dissolution state of the heated composition: o = dissolved. X = undissolved (residual).
Dissolution state of the composition after cooling: o = dissolved. X=undissolved (precipitated).
When lysine 2 hydrochloride is used, the composition obtained is in the form of a solution in which lysine 2 hydrochloride is completely dissolved, and the composition which is the object of the present invention is not obtained.
Claims (18)
1. A method for detecting microRNA in a biological sample, comprising the steps of:
(A) A step of mixing an acid with a biological sample to obtain a biological sample mixed solution;
(B) A step of storing the biological sample mixture obtained in the step (A);
(D) A step of recovering microRNA from the biological sample mixture obtained in the step (B); and
(E) Detecting the microRNA recovered in the step (D).
2. The detection method according to claim 1, wherein in the step (A), the pH of the biological sample mixture obtained is 2.0 to 4.0.
3. The detection method according to claim 1 or 2, comprising the following step (C) before step (D): the biological sample mixture obtained in the step (B) is mixed with a base, and the pH is adjusted to 6.0 to 9.0.
4. The detection method according to claim 3, wherein in the 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. The detection method according to any one of claims 1 to 5, wherein in the step (A), the acid used 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 in the step (A), the acid used is a homogeneous composition comprising a hydrochloride salt of a neutral amino acid, water, and an alcohol, and is a semisolid composition at 23 ℃.
8. The detection method according to any one of claims 1 to 7, wherein in the step (B), the biological sample mixture is stored for a period of time of 96 hours or less.
9. The detection method according to any one of claims 1 to 8, wherein in the step (B), the temperature of the biological sample mixture is kept at 1 to 30 ℃.
10. The detection method according to claim 3 or 4, wherein in the step (C), the base used is tris (hydroxymethyl) aminomethane, N- [ tris (hydroxymethyl) methyl ] glycine, triethanolamine, sodium hydroxide, potassium hydroxide, calcium hydroxide, N-bis (2-hydroxyethyl) glycine, N- (2-hydroxyethyl) piperazine-N' -2-ethane sulfonic acid or 2-hydroxy-3- [ tris (hydroxymethyl) methylamino ] -1-propane sulfonic acid.
11. The method according to any one of claims 1 to 10, wherein in the step (E), the method for detecting micrornas is a microarray method.
12. The detection method according to any one of claims 1 to 11, wherein the micrornas are micrornas used as disease markers.
13. The method of claim 12, wherein the disease is cancer or dementia.
14. A composition which is a homogeneous composition comprising a hydrochloride salt of a neutral amino acid, water and an alcohol, which is used as an acid in the step (A) of the detection method according to claim 1, and which is semi-solid at 23 ℃.
15. The composition of claim 14, wherein the weight ratio of the hydrochloride salt of the neutral amino acid in the composition is 65wt% or more and 70wt% or less, and the weight ratio of the alcohol is 2wt% or more and 6wt% or less.
16. The composition of claim 14 or 15, wherein the hydrochloride salt of the neutral amino acid 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.
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| JP2021-054811 | 2021-03-29 | ||
| JP2021-122220 | 2021-07-27 | ||
| JP2021122220 | 2021-07-27 | ||
| PCT/JP2022/012206 WO2022209943A1 (en) | 2021-03-29 | 2022-03-17 | Method for detecting microrna in biological sample, composition of hydrochloride of neutral amino acid, and container |
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