RNA storage method

Abstract

To provide a method for stably storing skin surface lipid-derived RNA.SOLUTION: A method for preventing degradation of skin surface lipid-derived RNA, comprises treating a sampled medium including skin surface lipids with an aqueous solution that contains at least one selected from the group consisting of guanidine hydrochloride and guanidinium thiocyanate each using water or an alcohol aqueous solution as a solvent.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for preserving RNA. [Background technology]

[0002] By analyzing nucleic acids derived from living organisms, such as DNA and RNA, various information including the physiological state of the living organism can be obtained. Nucleic acids derived from living organisms can be extracted from samples derived from living organisms, such as cells, tissues, body fluids, and secretions. Patent Document 1 describes that RNA is contained in skin surface lipids (SSLs), and that RNA derived from the SSLs can be used for analyzing living organisms. However, since the amount of RNA contained in SSLs is very small, it is necessary to extract RNA from SSLs collected from living organisms in as high a yield as possible.

[0003] Among nucleic acids derived from living organisms, RNA is more unstable and easily decomposed than DNA. Furthermore, ribonuclease (RNase), an RNA decomposing enzyme, is widely present not only in cells and tissues, but also in body fluids such as sweat and saliva, and in the environment. Conventionally, in order to prevent the decomposition of RNA in biological samples, measures such as immediately extracting RNA from collected samples, storing the RNA in the sample or extracted at -80°C, or using RNase inhibitors have been taken. For example, by adding a chaotropic salt such as a guanidine salt to a biological sample, proteins containing RNase in the sample can be denatured and RNase can be inactivated. However, even when a chaotropic salt is added, the decomposition of RNA cannot be completely prevented at ambient temperature, so that the RNA in the sample or extracted is usually stored at -80°C.

[0004] Patent Document 2 describes a simple method for extracting nucleic acids, which comprises the steps of adding a protein denaturing agent such as guanidine thiocyanate, guanidine hydrochloride, potassium thiocyanate, sodium thiocyanate, or urea to a biological sample to destroy cell membranes, cell walls, etc., and denaturing the proteins to liberate only the nucleic acids, adding an alcohol to aggregate the liberated nucleic acids, passing the resulting nucleic acid-containing extract through a filter to collect the nucleic acids on the filter, and solubilizing the nucleic acids with purified water or a low-concentration buffer solution and recovering them from the filter. Patent Document 3 describes that RNA samples from milk can be stored for long periods of time, for three days or more, by extracting RNA from milk using an RNA extraction agent containing phenol and guanidine thiocyanate and then quickly freezing the sample at -80°C. Patent Document 4 describes a method for extracting and storing nucleic acids from a sample, which comprises applying a solid matrix in which a composition containing a protein denaturant such as guanidinium hydrochloride, guanidinium thiocyanate, arginine, SDS, or urea, a reducing agent, and a buffer is present in a dry state, collecting nucleic acids from a sample, drying the solid matrix, and storing the nucleic acids in a dry state on the solid matrix under ambient conditions. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2018 / 008319 [Patent Document 2] JP 2009-153467 A [Patent Document 3] JP 2000-264898 A [Patent Document 4] JP 2018-68303 A Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention relates to a method for preventing degradation of RNA contained in surface skin lipids (SSL) collected from a subject and for stably storing the RNA. [Means for solving the problem]

[0007] The inventors have discovered that by collecting an SSL from a subject using a collection medium and then treating the collection medium containing the SSL with an aqueous solution containing at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate, with water or an aqueous alcohol solution as the solvent, it is possible to prevent degradation of the RNA contained in the SSL and to stably store the RNA.

[0008] Therefore, the present invention provides a method for preventing degradation of RNA derived from lipids on the skin surface, which comprises treating a collection medium containing lipids on the skin surface with an aqueous solution containing at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate, with water or an aqueous alcohol solution as a solvent. The present invention also provides a method for preserving RNA derived from lipids on the skin surface, comprising the steps of: Treating a collection medium containing lipids on the skin surface with an aqueous solution containing at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate, the aqueous solution being dissolved in water or an aqueous alcohol solution; and storing the treated collection medium; The present invention provides a method comprising: The present invention also relates to a collection medium for collecting lipids on the skin surface, and The kit for collecting RNA derived from lipids on the skin surface is provided, which includes an aqueous solution containing at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate, with water or an aqueous alcohol solution as a solvent, for treating the collection medium. The present invention also provides a method for recovering RNA derived from lipids on the skin surface of a subject, comprising the steps of: Providing a kit for collecting RNA derived from lipids on the skin surface to a subject who needs or desires to collect RNA derived from lipids on the skin surface, the kit comprising a collection medium for collecting lipids on the skin surface, and an aqueous solution containing at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate, the aqueous solution being dissolved in water or an aqueous alcohol solution, for treating the collection medium; and and recovering the collection medium from the subject, the collection medium containing the collected skin surface lipids from the subject; wherein the collection medium containing the collected skin surface lipids has been treated with the aqueous solution according to the method for preventing decomposition or preserving RNA derived from skin surface lipids, and has been stored at ambient temperature after collecting the skin surface lipids of the subject. A method is provided. Effect of the Invention

[0009] According to the method of the present invention, it is possible to prevent the degradation of RNA contained in SSL collected from a subject and to stably store the RNA. According to the present invention, it is possible to store RNA that is easily degraded and difficult to handle at ambient temperature. Furthermore, according to the present invention, it is possible to improve the yield of RNA from SSL and the accuracy of various analyses using the RNA. [Brief description of the drawings]

[0010] [Figure 1] Figure 1 shows an example of a container for storing a collection medium used in the kit for collecting SSL-derived RNA of the present invention, A: side view (left) and its cross-sectional view (right), B: top oblique view (left) and its cross-sectional view (right). [Diagram 2] FIG. 2 shows an example of the container of FIG. 1 containing a collection medium containing SSL-derived RNA and a desiccant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] All patents, non-patent publications, and other publications cited herein are hereby incorporated by reference in their entirety.

[0012] As used herein, "RNA" includes single-stranded RNA and double-stranded RNA, such as mRNA, tRNA, rRNA, small RNA (e.g., microRNA (miRNA), small interfering RNA (siRNA), PIWI-interacting RNA (piRNA)), long intergenic non-coding (linc) RNA, and the like.

[0013] As used herein, "skin surface lipids (SSL)" refers to the fat-soluble fraction present on the surface of the skin, also known as sebum. Generally, SSL mainly contains secretions secreted from exocrine glands such as sebaceous glands in the skin, and exists on the skin surface in the form of a thin layer covering the skin surface.

[0014] In this specification, unless otherwise specified, "skin" is a general term for an area including tissues such as the epidermis, dermis, hair follicles, sweat glands, sebaceous glands and other glands on the body surface.

[0015] In this specification, the term "ambient temperature" refers to the temperature in an environment where SSL can be collected, and preferably refers to room temperature or lower. Here, room temperature refers to a temperature of 1 to 30° C. as defined by the Japanese Pharmacopoeia.

[0016] In one aspect, the present invention provides a method for preventing degradation of SSL-derived RNA and a method for preserving SSL-derived RNA, which comprises treating a collection medium containing SSL collected from a subject with an aqueous solution containing at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate, the solvent of which is water or an aqueous alcohol solution.

[0017] In the method of the present invention, the subject from which SSL is collected may be any organism having SSL on the skin. Examples of subjects include mammals, including humans and non-human mammals, and preferably humans. For example, the subject may be a human or non-human mammal that requires or desires the analysis of its own nucleic acid. Alternatively, the subject may be a human or non-human mammal that requires or desires gene expression analysis in the skin, or the analysis of the condition of the skin or a site other than the skin using nucleic acid.

[0018] The site of skin from which the subject's SSL is collected includes, but is not limited to, skin from any part of the body, such as the head, face, neck, trunk, hands, and feet, for example, healthy skin, skin with diseases such as atopy, acne, dryness, inflammation (redness), and tumors, and skin with wounds.

[0019] The collection medium used to collect SSL from the skin of a subject includes SSL absorbent materials. Examples of SSL absorbent materials include materials that have a fibrous or porous structure and can collect and hold SSL in the voids in the structure by capillary action, materials that can adsorb SSL by a lipophilic material, and materials that combine both of these properties. Examples of materials for the SSL absorbent material include plastics such as polypropylene, synthetic fibers such as pulp, cellulose, cotton, and rayon, and preferably polypropylene with a porous structure. From the viewpoint of the collection efficiency of SSL and the efficiency of drying the collection medium described later, it is more preferable that the collection medium has a shape with a larger surface area to volume ratio. Preferably, the collection medium is in the form of a sheet. Specific examples of the collection medium include porous plastic films, paper, gauze, cotton balls, etc., or commercially available oil blotting paper or oil blotting film can be used. Preferred examples of the collection medium include porous plastic films having a thickness of 5 to 200 μm, a porosity of 5 to 50%, and a pore size of 0.2 to 5 μm, and cosmetic oil blotting sheets made of porous stretched films of plastic materials described in Japanese Patent No. 3055778 (for example, oil clear film made of porous polypropylene film by 3M (Hakugen Earth Co., Ltd.)). The porous plastic film may be hydrophilized as long as it does not inhibit the absorption of SSL.

[0020] In order to improve the collection efficiency of SSL, a collection medium that has been previously impregnated with a highly lipid-soluble solvent may be used. On the other hand, if the collection medium contains a highly water-soluble solvent or water, the adsorption of SSL is inhibited, which is not preferable. The collection medium is preferably used in a dry state.

[0021] In the method of the present invention, the collection medium containing SSL collected from a subject is treated with an aqueous solution containing at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate (hereinafter referred to as a treatment aqueous solution). Preferably, the treatment aqueous solution contains alcohol. Preferably, the treatment aqueous solution contains guanidine hydrochloride. The treatment aqueous solution can be prepared by dissolving at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate in a solvent containing water or an aqueous solution containing alcohol. Examples of alcohol that can be contained in the alcohol aqueous solution as the solvent include one or more selected from ethanol, isopropanol, butanol, etc., and ethanol is preferred from the viewpoint of efficiency of drying the collection medium described below. The alcohol concentration in the alcoholic aqueous solution as the solvent is not particularly limited, but from the viewpoint of the permeability to the SSL absorbent material and the efficiency of drying the collection medium described later, it is preferably 20% by volume or more, more preferably 30% by volume or more, and even more preferably 50% by volume or more, while from the viewpoint of the safety of handling the aqueous solution, it is preferably 70% by volume or less. Preferably, the alcohol concentration in the alcoholic aqueous solution as the solvent is 20% by volume or more and 70% by volume or less, more preferably 30% by volume or more and 70% by volume or less, even more preferably 50% by volume or more and 70% by volume or less, and even more preferably 50% by volume or more and less than 60% by volume. Here, the volume means the volume at 25°C.

[0022] The content of at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate in the aqueous treatment solution (the total amount of guanidine hydrochloride and guanidine thiocyanate) is preferably 0.50 g / mL or more, more preferably 0.55 g / mL or more, from the viewpoint of preventing RNA degradation, but may be any amount as long as it is equal to or less than the saturation concentration for the solvent of the aqueous treatment solution.

[0023] In the treatment of the collection medium with the aqueous treatment solution, the SSL contained in the collection medium is brought into contact with the aqueous treatment solution. For example, the treatment includes dropping the aqueous treatment solution onto the collection medium, applying the aqueous treatment solution onto the collection medium, spraying the aqueous treatment solution onto the collection medium, contacting the collection medium with a support that holds the aqueous treatment solution and permeating the collection medium with the aqueous treatment solution via the support, or immersing a part of the collection medium in the aqueous treatment solution. The aqueous treatment solution is permeated into the collection medium by these means. The aqueous treatment solution may be permeated into the entire collection medium, but it is sufficient that the aqueous treatment solution is permeated into at least the part of the collection medium that contains the SSL.

[0024] The amount of the aqueous treatment solution used to treat the collection medium is preferably an amount that is sufficient to cover at least the portion of the collection medium that contains SSL. For example, when the collection medium is a sheet-shaped collection medium, the amount of the aqueous treatment solution used is preferably an amount that covers 80% or more of the sheet area. In addition, the amount of the aqueous treatment solution is preferably an amount that can be retained in or on the collection medium. If the amount of the aqueous treatment solution applied is too small, RNA will be decomposed in the portion where the solution does not cover. On the other hand, if the amount of the aqueous treatment solution applied is too large, the SSL contained in the collection medium may flow out of the collection medium together with the aqueous treatment solution. In addition, if the amount of the aqueous treatment solution applied is too large, the collection medium treated with the aqueous treatment solution may take a long time to dry in the drying process described below, or a sufficient dry state may not be obtained. In light of these, the amount of the aqueous treatment solution applied to the collection medium may be appropriately adjusted according to the type and size of the collection medium.

[0025] Preferably, the method of the present invention further includes drying the collection medium treated with the treatment aqueous solution. By drying the collection medium, RNA degradation by RNase, a hydrolytic enzyme, is more effectively prevented. The means for drying the collection medium is not particularly limited as long as it can place the collection medium in a dry environment, but from the viewpoint of preventing RNase contamination, it is preferable to place the collection medium in a dry environment in a closed space system such as a container. Specifically, by coexisting the collection medium and a desiccant in a closed space system such as a container in which the collection medium can be stored, the drying of the collection medium can be promoted and RNA degradation can be more effectively prevented. The size and shape of the container are not particularly limited as long as it is made of a material that does not transmit moisture, can store the collection medium and the desiccant, and can be sealed. In addition, considering the case where the treatment aqueous solution contained in the collection medium comes into contact with the desiccant and generates heat, it is preferable that the container has a structure or configuration in which the collection medium and the desiccant do not come into direct contact with each other. Examples of desiccant include crystalline zeolite (molecular sieve), activated carbon, and silica gel. It is preferable to adjust the amount of desiccant used so that the closed space containing the collection medium can be kept in a dry environment (preferably by lowering the humidity to 0%). For example, 3 It is preferable to use 0.05 to 0.10 g of molecular sieve per cm2 of sheet area. 2 It is preferable to use 0.05 to 0.20 g of molecular sieve per 1 μL of the treatment aqueous solution that has been permeated into the collection medium, or 0.02 to 0.10 g of molecular sieve per 1 μL of the treatment aqueous solution that has been permeated into the collection medium.

[0026] The collection medium obtained by the above procedure, which has been treated with the aqueous treatment solution and preferably further dried, can be stored as a sample containing SSL-derived RNA. The above-mentioned steps of treatment with the aqueous treatment solution and drying prevent the SSL-derived RNA contained in the collection medium from being decomposed, so the resulting collection medium can be stored at ambient temperature or higher (e.g., 37°C or lower, 40°C or lower, 60°C or lower).

[0027] In another aspect, the present invention provides a kit for collecting SSL-derived RNA. The kit of the present invention includes the above-mentioned collection medium for collecting SSL, and an aqueous solution containing at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate in the above-mentioned water or alcohol aqueous solution as a treatment aqueous solution for treating the collection medium. In one embodiment, the kit further includes a container for storing the collection medium containing SSL after treatment with the treatment aqueous solution. The container can be sealed and can be used for drying or storing the collection medium after treatment with the treatment aqueous solution. Preferably, the container of the kit is used in combination with a desiccant. Therefore, preferably, the kit includes the container and a desiccant. For example, the collection medium after treatment with the treatment aqueous solution is placed in the container and sealed together with the desiccant stored inside the container, thereby promoting drying of the collection medium in the container. The dried collection medium can be stored as it is in the container. The container containing the collection medium can be stored at ambient temperature or higher (e.g., 37°C or lower, 40°C or lower, 60°C or lower).

[0028] FIG. 1 shows an example of a container for storing a collection medium used in the kit of the present invention. In FIG. 1, A shows a side view (left) and its cross-sectional view (right), and B shows an upper perspective view (left) and its cross-sectional view (right). The container is composed of a container cap 1A and a container body 1B, and at a portion 1-a where the cap 1A and the container body 1B contact each other, a thread is provided on the inner surface of the cap 1A and the outer surface of the container body 1B, and the cap 1A and the container body 1B are screwed together to form an enclosed space inside the container. The inside of the container is divided into a storage chamber 2 and a storage chamber 3 by a member 1C made of a breathable material or structure, and each storage chamber stores a collection medium after SSL collection that has been treated with a processing aqueous solution and a desiccant, respectively, so that the two can be stored in the same enclosed space inside the container without contacting each other. In the container, it is preferable that the desiccant is stored in the container beforehand. FIG. 2 shows an example of the container of FIG. 1 in a state in which a collection medium containing SSL-derived RNA and a desiccant are stored. A desiccant 4 is stored in storage chamber 3 inside the container, and collection medium 5 after SSL collection, which has been treated with a treatment aqueous solution, is stored in storage chamber 2 separated by breathable member 1C. The entire sealed space inside the container is placed in a dry environment via breathable member 1C, accelerating the drying of the collection medium, and RNA can be stored at ambient temperature or higher (for example, 37°C or lower, 40°C or lower, 60°C or lower).

[0029] The collection medium treated by the method of the present invention can be used as a sample containing SSL-derived RNA. RNA can be extracted from the collection medium according to a conventional procedure for RNA extraction from a sample. For example, RNA can be extracted from the collection medium according to the phenol / chloroform method and its modifications, such as the PCI (Phenol / Chloroform / Isoamyl alcohol) method, the AGPC (acid guanidinium thiocyanate-phenol-chloroform extraction) method, and the AGPC modification in which guanidine thiocyanate and phenol are mixed in advance. Alternatively, RNA can be extracted from the collection medium using a commercially available RNA extraction reagent (e.g., TRIzol (registered trademark) Reagent, QIAzol Lysis Reagent, ISOGEN, RNeasy Mini Kit, etc.).

[0030] The extracted RNA is the subject's SSL-derived RNA and can be used for various analyses. For example, the mRNA contained in the SSL-derived RNA can be converted to cDNA using an Oligo(dT) primer and then used for gene expression analysis, transcriptome analysis, etc. Alternatively, by examining the presence or absence of a target RNA in the subject's SSL-derived RNA, it is possible to perform functional analysis of the subject, diagnosis of a disease, and evaluation of the efficacy of a drug administered to the subject.

[0031] In another aspect, the present invention provides a method for collecting RNA derived from lipids on the skin surface of a subject. The method of the present invention includes providing a kit for collecting RNA derived from lipids on the skin surface of the present invention to a subject who needs or desires to collect RNA derived from lipids on the skin surface, and collecting the collection medium containing lipids on the skin surface collected from the subject using the collection medium provided in the kit. Here, the collection medium containing lipids on the skin surface collected from the subject has been treated with the aqueous solution according to the method for preventing degradation or preserving RNA derived from lipids on the skin surface of the present invention. In addition, the collection medium containing lipids on the skin surface collected from the subject has been stored at ambient temperature or higher (e.g., 37°C or lower, 40°C or lower, 60°C or lower) after collecting lipids on the skin surface of the subject. Here, the method of providing a kit for collecting RNA derived from lipids on the skin surface and the method of recovering the collection medium from the subject after collecting lipids on the skin surface are not particularly limited, and any known transportation means can be used, such as a mail service, a home delivery service by a freight carrier, receipt of the collection kit by the subject or a representative at a distribution center, or bringing of the collection medium to a collection center by the subject or a representative.

[0032] As exemplary embodiments of the present invention, the following substances, manufacturing methods, uses, methods, etc. are further disclosed in the present specification. However, the present invention is not limited to these embodiments.

[0033] [1] A method for preventing the degradation of RNA derived from lipids on the surface of the skin, comprising treating a collection medium containing lipids on the surface of the skin with an aqueous solution containing at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate, with water or an aqueous alcohol solution as a solvent. [2] A method for preserving RNA derived from lipids on the skin surface, comprising: Treating a collection medium containing lipids on the skin surface with an aqueous solution containing at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate, the aqueous solution being dissolved in water or an aqueous alcohol solution; and storing the treated collection medium; A method comprising: [3] The method according to [1] or [2], preferably further comprising drying the collection medium treated with the aqueous solution. [4] Preferably, the solvent is an aqueous alcohol solution, and the concentration of alcohol in the aqueous alcohol solution is: Preferably, it is 20% by volume or more. More preferably, it is 20% by volume or more and 70% by volume or less. More preferably, it is 30% by volume or more. More preferably, the content is 30% by volume or more and 70% by volume or less. More preferably, it is 50% by volume or more. More preferably, the content is 50% by volume or more and 70% by volume or less. More preferably, it is 50% by volume or more and less than 60% by volume. The method according to any one of [1] to [3]. [5] The concentration of at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate in the aqueous solution is Preferably, it is 0.50 g / mL or more. More preferably, it is 0.55 g / mL or more. The method according to any one of [1] to [4]. [6] The treatment of the collection medium containing the lipids on the skin surface with the aqueous solution Preferably, the method includes contacting the aqueous solution with the lipids on the skin surface contained in the collection medium, More preferably, the method comprises permeating the collection medium with the aqueous solution; More preferably, the aqueous solution is permeated into at least a portion of the collection medium containing lipids on the skin surface. The method according to any one of [1] to [5]. [7] The collection medium is Preferably, the material is a lipid-absorbent material on the surface of the skin. More preferably, it is a porous plastic film. The method according to any one of [1] to [6]. [8] The method according to any one of [1] to [7], wherein the alcohol is at least one selected from the group consisting of ethanol, isopropanol, and butanol. [9] Drying the collection medium treated with the aqueous solution Preferably, the method comprises placing the collection medium in a closed space system under a dry environment; More preferably, the method includes coexisting the collection medium and a desiccant in a container. The method according to any one of [3] to [8].

[10] The method according to any one of [2] to [9], wherein storing the collection medium treated with the aqueous solution includes storing the collection medium at a temperature of preferably 60°C or less, more preferably 40°C or less, even more preferably 37°C or less, even more preferably 1 to 60°C, even more preferably 1 to 40°C, even more preferably 1 to 37°C, and even more preferably 1 to 30°C.

[0034]

[11] A collection medium for collecting lipids on the skin surface, and The kit for collecting RNA derived from lipids on the skin surface comprises an aqueous solution containing at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate, with water or an aqueous alcohol solution as a solvent, for treating the collection medium.

[12] The kit according to

[11] , preferably further comprising a container and a desiccant for storing a collection medium containing the lipids on the skin surface after treatment with the aqueous solution.

[13] The kit according to

[12] , wherein the container preferably contains the desiccant therein.

[14] Preferably, the solvent is an aqueous alcohol solution, and the concentration of alcohol in the aqueous alcohol solution is: Preferably, it is 20% by volume or more. More preferably, it is 20% by volume or more and 70% by volume or less. More preferably, it is 30% by volume or more. More preferably, the content is 30% by volume or more and 70% by volume or less. More preferably, it is 50% by volume or more. More preferably, the content is 50% by volume or more and 70% by volume or less. More preferably, it is 50% by volume or more and less than 60% by volume. The kit according to any one of

[11] to

[13] .

[15] The concentration of at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate in the aqueous solution is Preferably, it is 0.50 g / mL or more. More preferably, it is 0.55 g / mL or more. The kit according to any one of

[11] to

[14] .

[16] The collection medium is Preferably, the material is a lipid-absorbent material on the surface of the skin. More preferably, it is a porous plastic film. The kit according to any one of

[11] to

[15] .

[17] The kit according to any one of

[11] to

[16] , wherein the alcohol is at least one selected from the group consisting of ethanol, isopropanol, and butanol.

[18] The container comprises: Preferably, the container is a sealable container, More preferably, the container is as shown in FIG. The kit according to any one of

[12] to

[17] .

[0035]

[19] A method for recovering RNA derived from lipids on the skin surface of a subject, comprising: Providing a kit for collecting RNA derived from lipids on the skin surface according to any one of

[11] to

[18] above to a subject who needs or desires to collect RNA derived from lipids on the skin surface, the kit comprising a collection medium for collecting lipids on the skin surface, and an aqueous solution containing at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate, the aqueous solution being dissolved in water or an aqueous alcohol solution, for treating the collection medium; and and recovering the collection medium from the subject, the collection medium containing the collected skin surface lipids from the subject; wherein the collection medium containing the collected skin surface lipids has been treated with the aqueous solution according to the method for preventing degradation or preserving RNA derived from skin surface lipids described in any one of [1] to

[10] , and has been stored at ambient temperature after collecting the skin surface lipids of the subject. method. EXAMPLES

[0036] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0037] Example 1 Prevention of RNA degradation by guanidine salts (1) Preparation of guanidine hydrochloride solution and guanidine thiocyanate solution Guanidine hydrochloride (FUJIFILM Wako Pure Chemical Industries, Ltd.) was dissolved in a 57% (v / v) aqueous ethanol solution to a concentration of 6 M. Guanidine thiocyanate (FUJIFILM Wako Pure Chemical Industries, Ltd.) was dissolved in a 57% (v / v) aqueous ethanol solution to a concentration of 6 M.

[0038] (2) Preparation of oil blotting film Using four oil blotting films (Oil Clear Film, Shiraki Earth Co., Ltd., 3M porous polypropylene film, 25 x 40 mm), SSL was collected from the entire face of the subject. Separately, RNA was extracted from human skin sections using RNeasy Mini Kit (Qiagen) and dissolved in 57% (v / v) ethanol aqueous solution so that the RNA concentration was 1 μg / mL. The obtained human skin-derived RNA solution was evenly applied to the entire surface of each of the four oil blotting films in an amount of 20 μL each, and allowed to permeate. Of the four oil blotting films to which RNA was applied, one was evenly applied with the guanidine hydrochloride solution prepared in (1), and the other was evenly applied with the guanidine thiocyanate solution prepared in (1), each in an amount of 20 μL each, and allowed to permeate. The remaining two oil blotting films were not applied with the guanidine salt solution. The four oil blotting films were each placed into a screw tube (volume 20 mL) and stored at -80°C or room temperature (18 to 25°C) for four days (Table 1).

[0039] (3) Evaluation of RNA degradation prevention effect RNA was extracted from the oil-blotting film after storage prepared in (2), the extracted RNA was converted to cDNA by reverse transcription, and then the cDNA was subjected to PCR, and the reaction product was purified. Specifically, RNA was extracted from the oil-blotting film using RNeasy Mini Kit (Qiagen), and the obtained RNA was reverse transcribed and converted to cDNA using SuperScript VILO cDNA Synthesis kit (Thermo Scientific). The cDNA was subjected to PCR using Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies Japan), and the obtained reaction product was purified using magnetic beads (Ampure XP: Beckman Coulter), to obtain 10 μL of cDNA purified solution. The cDNA concentration in the purified solution was quantified using Agilent 4200 TapeStation (Agilent Technologies), and the relative cDNA concentration of each sample to the cDNA concentration of the sample stored at -80°C was determined. As shown in Table 1, RNA decomposition at room temperature was prevented by guanidine salt treatment. Furthermore, guanidine hydrochloride was more effective at preventing RNA degradation than guanidine thiocyanate.

[0040] [Table 1]

[0041] Example 2 Prevention of RNA degradation by drying (1) Preparation of desiccant Three screw tubes (capacity 20 mL) were prepared, and molecular sieves 13X 1 / 8 (FUJIFILM Wako Pure Chemical Industries, Ltd.) were added to one of them so that the ratio of screw tube volume (mL) to molecular sieves mass (g) was 20: 1. Specifically, 1.0 g of molecular sieves was added to a 20 mL screw tube.

[0042] (2) Preparation of oil blotting film SSL was collected from the entire face of the subject using three oil blotting films (oil clear film, Hakugen Earth Co., Ltd., 25 x 40 mm). 20 μL of the human skin-derived RNA solution prepared in Example 1 (2) was evenly applied to each of the three oil blotting films and allowed to penetrate. Each of the three oil blotting films was placed in the screw tube prepared in (1) and stored at -80°C or room temperature (18 to 25°C) for three days (Table 2).

[0043] (3) Evaluation of RNA degradation prevention effect RNA was extracted from the oil blotting film after storage prepared in (2) in the same manner as in Example 1 (3), converted to cDNA and subjected to PCR, the reaction products were purified and quantified, and the relative cDNA concentration of each sample was determined in comparison with the cDNA concentration of the sample stored at -80°C. As shown in Table 2, drying prevented RNA decomposition at room temperature.

[0044] [Table 2]

[0045] Example 3 Prevention of RNA degradation by guanidine salts and drying (1) Preparation of guanidine hydrochloride solution A guanidine hydrochloride solution was prepared in the same manner as in Example 1(1).

[0046] (2) Preparation of desiccant Three screw tubes were prepared in the same manner as in Example 2(1), and a desiccant was added to one of them.

[0047] (3) Preparation of oil blotting film SSL was collected from the entire face of the subject using three oil blotting films (oil clear film, Hakugen Earth Co., Ltd., 25 x 40 mm). 20 μL of the human skin-derived RNA solution prepared in Example 1 (2) was evenly applied to each of the three oil blotting films and allowed to penetrate. Of the three oil blotting films to which the RNA was applied, 20 μL of the guanidine hydrochloride solution prepared in (1) was evenly applied to the entire film and allowed to penetrate. The film to which the guanidine hydrochloride solution was applied was placed in the screw tube prepared in (2) to which the desiccant was added. The remaining two oil blotting films were not coated with the guanidine hydrochloride solution and were each placed in the screw tube prepared in (2) to which the desiccant was not added. Each sample was stored at -80°C or room temperature (18 to 25°C) for 3 days (Table 3).

[0048] (4) Evaluation of RNA degradation prevention effect RNA was extracted from the oil blotting film after storage prepared in (3) in the same manner as in Example 1(3), converted to cDNA and subjected to PCR, the reaction products were purified and quantified, and the relative cDNA concentration of each sample was determined in comparison with the cDNA concentration of the sample stored at -80°C. As shown in Table 3, the combined use of guanidine salt treatment and drying significantly prevented RNA decomposition at room temperature.

[0049] [Table 3]

[0050] Example 4 Prevention of RNA decomposition by guanidine salt aqueous solution or guanidine salt-alcohol aqueous solution and drying (1) Preparation of guanidine hydrochloride solution Guanidine hydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 30% (v / v) aqueous ethanol solution or water (UltraPure DNase / RNase-Free Distilled Water; Thermo Fisher Scientific KK) to a concentration of 6 M.

[0051] (2) Preparation of desiccant Four screw tubes (capacity 20 mL) were prepared, and molecular sieves 13X 1 / 8 (FUJIFILM Wako Pure Chemical Industries, Ltd.) were added to two of them so that the ratio of screw tube volume (mL) to molecular sieves mass (g) was 20: 1. Specifically, 1.0 g of molecular sieves was added to a 20 mL screw tube.

[0052] (3) Preparation of oil blotting film Using four oil blotting films (sweat-removing oil clear film, Hakugen Earth Co., Ltd., 3M's porous polypropylene film, 25 x 40 mm), SSL was collected from the entire face of the subject. Separately, Human HeLa Cell Total RNA (Takara Bio Inc.) was prepared with UltraPure DNase / RNase-Free Distilled Water (Thermo Fisher Scientific KK) using RNeasy Mini Kit (Qiagen) so that the RNA concentration was 1 μg / mL. The obtained HeLa Cell-derived RNA solution was evenly applied to each of the four oil blotting films in an amount of 20 μL each over the entire film and allowed to penetrate. Of the four oil blotting films to which RNA was applied, one was evenly applied with 20 μL of the guanidine hydrochloride solution in a 30% (v / v) ethanol aqueous solution prepared in (1) as a solvent, and the other was evenly applied with 20 μL of the guanidine hydrochloride solution in a water solvent as a solvent, over the entire film, and allowed to penetrate. The two films coated with the guanidine hydrochloride solution were placed in the screw tubes prepared in (2) containing the desiccant. The remaining two oil removing films were not coated with the guanidine hydrochloride solution and were placed one by one in the screw tubes prepared in (2) containing no desiccant. Each sample was stored at -80°C or 37°C for 3 days (Table 4).

[0053] (4) Evaluation of RNA degradation prevention effect RNA was extracted from the oil blotting film after storage prepared in (3) in the same manner as in Example 1(3), converted to cDNA and subjected to PCR, the reaction products were purified and quantified, and the relative cDNA concentration of each sample was determined in comparison with the cDNA concentration of the sample stored at -80° C. As shown in Table 4, RNA decomposition during storage at 37° C. was significantly prevented by guanidine salt treatment and drying.

[0054] [Table 4]

[0055] Example 5 Prevention of RNA degradation by guanidine salt and drying during storage at 60°C (1) Preparation of guanidine hydrochloride solution A guanidine hydrochloride solution was prepared in the same manner as in Example 1(1).

[0056] (2) Preparation of desiccant Three screw tubes were prepared in the same manner as in Example 2(1), and a desiccant was added to one of them.

[0057] (3) Preparation of oil blotting film SSL was collected from the entire face of the subject using three oil blotting films (oil clear film, Hakugen Earth Co., Ltd., 25 x 40 mm). 20 μL of the HeLa Cell-derived RNA solution prepared in Example 4 (3) was evenly applied to each of the three oil blotting films and allowed to penetrate. Of the three oil blotting films coated with RNA, 20 μL of the guanidine hydrochloride solution prepared in (1) was evenly applied to the entire film and allowed to penetrate. The film coated with the guanidine hydrochloride solution was placed in the screw tube prepared in (2) to which a desiccant had been added. The remaining two oil blotting films were not coated with the guanidine hydrochloride solution and were each placed in the screw tube prepared in (2) to which a desiccant had not been added. Each sample was stored at -80°C or 60°C for 3 days (Table 5).

[0058] (4) Evaluation of RNA degradation prevention effect RNA was extracted from the oil blotting film after storage prepared in (3) in the same manner as in Example 1(3), converted to cDNA and subjected to PCR, the reaction products were purified and quantified, and the relative cDNA concentration of each sample was determined in comparison with the cDNA concentration of the sample stored at -80° C. As shown in Table 5, RNA decomposition during storage at 60° C. was significantly prevented by guanidine salt treatment and drying.

[0059] [Table 5]

Claims

1. A method for preventing degradation of RNA derived from lipids on the skin surface, comprising: treating a sheet-shaped skin surface lipid-absorbing material containing skin surface lipids with an aqueous solution containing at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate, the aqueous solution being water or an aqueous alcohol solution; and drying the sheet-like lipid-absorbent material on the skin surface that has been treated with the aqueous solution. A method comprising:

2. A method for preserving RNA derived from lipids on the skin surface, comprising: treating a sheet-shaped skin surface lipid-absorbing material containing skin surface lipids with an aqueous solution containing at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate, the aqueous solution being water or an aqueous alcohol solution; Furthermore, the lipid-absorbent material on the sheet-like skin surface treated with the aqueous solution is dried; and storing the dried sheet-like lipid-absorbent material on the skin surface; A method comprising:

3. 3. The method according to claim 1, wherein the solvent is an aqueous alcohol solution having an alcohol concentration of 20% by volume or more and 70% by volume or less.

4. The method according to any one of claims 1 to 3, wherein the concentration of the at least one selected from the group consisting of guanidine hydrochloride and guanidine thiocyanate in the aqueous solution is 0.50 g / mL or more.

5. The method according to any one of claims 1 to 4, wherein the treatment comprises permeating the aqueous solution into a sheet-shaped lipid-absorbent material on the surface of the skin, the lipid-absorbent material being on the surface of the skin.

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