WO2012039395A1 - small RNAの取得方法 - Google Patents
small RNAの取得方法 Download PDFInfo
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- WO2012039395A1 WO2012039395A1 PCT/JP2011/071379 JP2011071379W WO2012039395A1 WO 2012039395 A1 WO2012039395 A1 WO 2012039395A1 JP 2011071379 W JP2011071379 W JP 2011071379W WO 2012039395 A1 WO2012039395 A1 WO 2012039395A1
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
Definitions
- the present invention relates to a method for obtaining small RNAs suitable for high throughput.
- RNA small RNA
- piRNA piwi-interacting RNA
- a carrier carrying an anti-Argonaute family antibody such as anti-Ago2 antibody
- small RNA by obtaining an Argonaute family protein-small RNA complex and separating the small RNA from this complex (Patent Document 1).
- small RNA is extracted using a phenol / chloroform / isoamyl alcohol mixed solution or an acidic guanidine / phenol / chloroform mixed solution as a method for separating small RNA from an Argonaute family protein-small RNA complex bound to a carrier. Method is used.
- an object of the present invention is to provide a method that can be applied to high throughput and can easily and efficiently acquire small RNA.
- the present invention is directed to “contacting a carrier on which a substance having affinity for a small RNA binding protein is immobilized on a surface thereof and a complex of the small RNA binding protein and the small RNA (protein RNA complex), A protein RNA complex is bound to a carrier, and the carrier to which the protein RNA complex is bound is heated at 70 to 100 ° C. in water or a buffer solution having a pH of 3.0 to 8.0 to release small RNAs and obtained. Is a method for obtaining small RNAs.
- the method of the present invention does not require the extraction and precipitation operation or the use of a protein denaturing agent such as a surfactant, so the eluate can be directly used for reactions such as reverse transcription and PCR. It can be used and can be incorporated as a step of screening by a high-throughput method, and small RNA can be easily obtained.
- a magnetic material is used as the carrier, even if the carrier has a small particle size, there is no need for centrifugation after the immunoprecipitation reaction, and small RNA can be obtained more easily.
- the method of the present invention can also acquire small RNA with high efficiency equivalent to or higher than that of the conventional method with respect to the efficiency of acquiring small RNA. Therefore, according to the method of the present invention, even if the number of samples is large, small RNA can be obtained in a short time, so that this method is suitable for research, diagnosis, etc. that handle a large number of samples.
- FIG. 1 shows the amount of miRNA (miR-92a) recovered in various extraction methods after immunoprecipitation of human plasma with anti-human Ago2 antibody-immobilized beads obtained in Example 1, Comparative Example 1 and Comparative Example 2.
- FIG. From the left the results of recovery by SDS elution / phenol / chloroform extraction method, the results of recovery by acidic elution method, and the results of recovery by heat elution method of the present invention are shown respectively.
- Example 2 shows that human plasma obtained in Example 2 was immunoprecipitated with anti-human Ago2 antibody-immobilized beads, nuclease-free water was added, and 50 ° C., 60 ° C., 70 ° C., 80 ° C., 90 ° C., It is a figure showing the amount of miRNA (miR-92a) obtained by heating and elution at 100 ° C.
- FIG. 3 shows that human plasma obtained in Example 3 was immunoprecipitated with anti-human Ago2 antibody-immobilized beads, nuclease-free water was added, and each of 70 ° C., 80 ° C., 90 ° C., and 100 ° C. for 10 seconds.
- FIG. 4 is a view showing the amount of miRNA (miR-92a) obtained by heating and elution for 30 seconds, 60 seconds, 90 seconds, 120 seconds, and 180 seconds.
- FIG. 4 shows the amount of miRNA (miR-92a) obtained in Example 4 after immunoprecipitation of human plasma with anti-human Ago2 antibody-immobilized beads, various buffers added, and heated and eluted at 90 ° C. for 90 seconds.
- various buffers include nuclease-free water, 10 mM citrate buffer (pH 3.0), 10 mM citrate buffer (pH 4.0), 10 mM citrate buffer (pH 4.5), and 10 mM citrate buffer.
- FIG. 5 shows that human plasma obtained in Example 5 was immunoprecipitated with anti-human Ago2 antibody-immobilized beads, nuclease-free water with or without various nucleic acids added, and heated and eluted at 90 ° C. for 90 seconds. It is a figure showing the amount of obtained miRNA (miR-92a).
- FIG. 6 shows the miR-16 and miR-92a amounts recovered from human plasma by various methods obtained in Example 6, Comparative Example 3 and Comparative Example 4.
- the amount of miRNA when immunoprecipitated with nuclease-free water after immunoprecipitation with anti-human Ago2 antibody-immobilized beads, and nuclease-free water with E. coli ribosomal RNA added after immunoprecipitation with anti-human Ago2 antibody-immobilized beads (final concentration 1 ng) miRNA amount when heated and eluted with anti-human Ago2 antibody-immobilized beads after heat-precipitation with salmon sperm DNA-added nuclease-free water (final concentration 1 ng / ⁇ L)
- miRNA is eluted with 10 mM citrate buffer (pH 5.5) after immunoprecipitation with Ago2 antibody-immobilized beads, and then extracted with SDS / phenol / chloroform after immunoprecipitation with anti-human Ago2 antibody-immobilized beads Represents the miRNA amount when human plasma is extracted by AGPC.
- the small RNA according to the present invention is usually RNA of 5 to 200 nt, preferably 10 to 50 nt, more preferably 10 to 30 nt, and any protein that binds to it is present.
- 5S ribosomal RNA 5S ribosomal RNA
- tRNA transfer RNA
- snRNA small nuclear RNA
- snoRNA small nucleolar RNA
- tiRNA transcription ⁇ initiation RNA
- spliRNA small interfering RNA
- siRNA piwi-interacting RNA
- miRNA micro RNA
- miRNA micro RNA
- the small RNA binding protein according to the present invention varies depending on the type of target small RNA, but may be any protein that can bind to the small RNA according to the present invention.
- a ribosomal protein a small nuclear ribonucleoprotein (snRNP protein), small nucleolar ribonucleic acid protein (snoRNP protein), Argonatute family protein and the like, among which Argonaute family protein is preferable.
- Argonaute family proteins include Argonaute subfamily such as Ago1, AgoA2, Ago 3, and Ago 4 and Piwi subfamily such as Piwil 1, Piwil 2, Piwil 3, and Piwil 4. Among them, Argonaute A subfamily and the like are preferable, and Ago 2 is particularly preferable.
- the complex of the small RNA binding protein according to the present invention and the small RNA is the small RNA binding protein according to the present invention and the small RNA according to the present invention. Is a complex bound.
- the protein RNA complex according to the present invention includes those that further contain a protein (for example, TNRC6A, Gemin3, Gemin4, FMRP, etc.) that binds to the complex.
- Specific examples of the complex include, for example, RISC (RNA-induced silencing complex).
- a substance having an affinity for a small RNA binding protein according to the present invention (hereinafter sometimes abbreviated as a protein affinity substance according to the present invention) binds to a substance having an affinity for a small RNA binding protein.
- An antibody or an aptamer having an affinity (binding ability) for the small RNA-binding protein may be used, and an antibody is preferred, although it may vary depending on the type of the small RNA binding protein. Specifically, for example, when Ago2 is used as a small RNA-binding protein, an anti-Ago2 antibody is used, and when Ago3 is used, an anti-Ago3 antibody is used.
- the origin of the antibody used as the protein affinity substance according to the present invention is not particularly limited, and it may be either a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferred. These antibodies may be commercially available, or may be used as F (ab ′) 2 , Fab ′, or Fab after digestion with an enzyme such as pepsin or papain if necessary.
- immunized cells such as spleen cells and lymphocytes of animals such as rats and mice immunized with the measurement object as an immunogen, and myeloma cells and the like
- a hybridoma is prepared by fusing the cells having the property of permanently proliferating with a cell fusion technique (Nature, 256, 495, 1975) known per se developed by Keller and Milstein et al.
- a hybridoma that produces a specific monoclonal antibody is selected, and the hybridoma is cultured in a medium or administered into the abdominal cavity of an animal to produce antibodies in ascites, and the desired monoclonal antibody is produced from the culture or ascites
- the antibody having the above-mentioned properties is produced by a method known per se (Eur. J. Immunol., 6, 511, 1976). Cells were prepared for, and a method of harvesting the desired monoclonal antibodies are exemplified by culturing the cells.
- the aptamer production method the method described in US Pat. No. 5,270,163 is preferred.
- a carrier in a carrier in which a substance having an affinity for a small RNA binding protein according to the present invention is immobilized on its surface hereinafter sometimes abbreviated as a carrier according to the present invention
- an ordinary immunological method is used. Any carrier can be used as long as it is used in the measurement method.
- These carriers can be used in a wide variety of forms such as tubes, beads, disc-like pieces, and particles. Among them, it is preferable to use particles, and the size of the particles is not particularly limited, but it is preferable to use a particle size of several nm to 100 ⁇ m according to the purpose and application.
- the carrier according to the present invention also includes a carrier in which a layer containing a polymer compound is formed on the surface of the carrier as described above.
- a carrier rarely adsorbs non-specific proteins on the surface of the carrier. Since there is no need to add a blocking agent or the like used to prevent it, it is preferable.
- a polymer described in JP-A-2009-148243 can be used according to the method described in the publication.
- a vinyl group, an allyl group, a methacryl group, an epoxy group, a polymerizable functional group such as a styrene group, or a chain transfer group such as a mercapto group or an amino group is introduced on the carrier surface according to the present invention.
- the obtained carrier is mixed with an ethylenically unsaturated polymerizable monomer having a functional group for immobilizing at least the protein affinity substance according to the present invention, and if necessary, an ethylenically unsaturated polymerizable monomer further having an alkyleneoxy group.
- the polymerization reaction is allowed to proceed.
- it can be obtained as follows.
- silica beads are added to an acetic acid aqueous solution (for example, pH 2 to 4) of a silane coupling agent such as 0.01 to 1.0 mol / L of methacryloxypropyltrimethoxysilane, and 10 to 10 to 50 to 100 ° C. Reaction is performed for 180 minutes to produce silica beads having a polymerizable functional group or chain transfer group.
- a silane coupling agent such as 0.01 to 1.0 mol / L of methacryloxypropyltrimethoxysilane, and 10 to 10 to 50 to 100 ° C.
- Reaction is performed for 180 minutes to produce silica beads having a polymerizable functional group or chain transfer group.
- p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate and polyethylene glycol methyl ether methacrylate are mixed at, for example, 1:99 to 70:30, and dissolved in a solvent such as dehydrated ethanol.
- silica beads having a polymerizable functional group or chain transfer group is added.
- silica beads are added so that the above-mentioned polymer is present in an amount of 0.1 to 10 mmol, reacted at 50 to 80 ° C. for 10 to 30 hours, if necessary, in an argon atmosphere, and then dried to increase the surface.
- a carrier having a layer containing the molecular compound is obtained.
- the amount of the protein affinity substance according to the present invention immobilized on the carrier according to the present invention varies depending on the type of the protein affinity substance used, but is usually 0.1 to 10 mg, preferably 1 to 10 mg, per 1 g of the carrier. It is.
- the carrier according to the present invention in which a substance having affinity for a small RNA binding protein is immobilized on its surface (hereinafter abbreviated as the protein affinity substance-immobilized carrier according to the present invention) relates to the above-mentioned present invention.
- the protein affinity substance according to the present invention is immobilized on the surface of a carrier.
- Examples of the method for immobilizing the protein affinity substance according to the present invention on the surface of the carrier according to the present invention include known immobilization methods such as a method of immobilization by chemical bonds such as covalent bonds, and physical adsorption. And immobilization methods known per se such as a method of immobilizing by affinity binding via a substance having affinity for an antibody such as protein A or protein G, etc. However, a method of immobilizing by chemical bonding, a method of immobilizing by affinity bonding, and the like are preferable.
- a method of immobilizing by chemical bonding is preferable, and in particular, a functional group introduced on the particle surface.
- a method of immobilizing a protein affinity substance using a group is particularly preferred.
- the functional group introduced into the particle surface in accordance with the functional group in the protein affinity substance may be appropriately selected.
- the following may be performed as a specific method for binding the functional group introduced on the particle surface and the functional group in the protein affinity substance.
- a carboxyl group and a carboxyl group when bonded, they may be bonded using a polyhydroxy compound or an alkylene oxide as a condensing agent.
- -OQO- represents a group derived from a polyhydroxyl compound or an alkylene oxide.
- a polyamine compound may be used as a condensing agent.
- a hydroxyl group and a hydroxyl group may be bonded using polycarboxylic acid as a condensing agent.
- a hydroxyl group and a hydroxyl group may be bonded using alkylene oxide as a condensing agent.
- an isocyanate group and an isocyanate group may be bonded using a polyhydroxy compound as a condensing agent.
- an amino group and a carboxyl group When an amino group and a carboxyl group are bonded, they may be bonded using a dehydrating agent.
- a hydroxyl group and a carboxyl group may be bonded using a dehydrating agent.
- the method for immobilizing the protein affinity substance according to the present invention on the surface of the carrier according to the present invention is, for example, usually 2 ⁇ g / mL to 200 ⁇ g / mL of the protein affinity substance according to the present invention, preferably Usually, 0.5 mL of a solution containing 20 ⁇ g / mL to 200 ⁇ g / mL (protein affinity substance-containing solution according to the present invention) and 10 mg of the carrier according to the present invention are brought into contact with each other in the presence of an appropriate condensing agent if necessary.
- the reaction may be performed at 20 to 50 ° C., preferably 30 to 40 ° C., usually for 1 to 20 hours, preferably 1 to 10 hours, more preferably 2 to 5 hours.
- the condensing agent used may be appropriately selected from those used in conventional methods in this field, and the condensing agents described in the above-mentioned combination of functional groups are preferable.
- a blocking agent such as ethanolamine to inactivate the functional group on the carrier surface according to the present invention where the protein affinity substance is not immobilized. It is desirable.
- the solvent for preparing the protein affinity substance-containing solution according to the present invention may be any solvent as long as it does not prevent the protein affinity substance to be used from adsorbing or binding to the insoluble carrier.
- purified water such as pH Buffers having a buffering action at 5.0 to 10.0, preferably pH 8.5 to 10 (for example, phosphate buffer, Tris buffer, Good buffer, glycine buffer, borate buffer, sodium bicarbonate buffer, etc.) are preferable.
- the concentration of the buffer in these buffers is appropriately selected from the range of usually 0.1 to 5 ⁇ M, preferably 0.6 to 2.5 ⁇ M.
- this solution contains, for example, saccharides, salts such as NaCl, surfactants, preservatives, proteins, etc., as long as they do not prevent the antibody from adsorbing or binding to the insoluble carrier. Also good.
- the protein affinity substance-immobilized carrier according to the present invention obtained as described above may be subjected to a blocking treatment usually performed in this field.
- the protein affinity substance-immobilized carrier according to the present invention is specifically manufactured as follows when, for example, Ago2 is used as the protein affinity substance according to the present invention. That is, the protein affinity substance according to the present invention, such as anti-Ago2 antibody, is present in an amount of 0.1 to 10 mg per 1 g of the carrier according to the present invention as described above, and added to the sodium hydrogen carbonate buffer at 30 to 40 ° C. By reacting for 2 to 10 hours, the protein affinity substance according to the present invention is immobilized on the surface of the particles, and the protein affinity substance-immobilized carrier according to the present invention is obtained.
- Ago2 is used as the protein affinity substance according to the present invention. That is, the protein affinity substance according to the present invention, such as anti-Ago2 antibody, is present in an amount of 0.1 to 10 mg per 1 g of the carrier according to the present invention as described above, and added to the sodium hydrogen carbonate buffer at 30 to 40 ° C.
- the method for obtaining small RNA of the present invention includes: [Step (1)] The protein affinity substance-immobilized carrier according to the present invention and the protein RNA complex according to the present invention are contacted, and the protein RNA complex is bound to the carrier, [Step (2)] If necessary, the carrier to which the protein RNA complex is bound is washed, [Step (3)] The carrier to which the protein RNA complex is bound is heated at 70 to 100 ° C. in water or a buffer having a pH of 3.0 to 8.0 to release small RNA.
- RNA complex a solution containing a protein RNA complex and a protein affinity substance-immobilized carrier according to the present invention are mixed, and 2 to 37 ° C., preferably 2 to 10 ° C. for 1 to 30 hours, preferably 2 to 10 hours, More preferably, the reaction is performed for 2 to 5 hours, and the protein RNA complex according to the present invention is bound to the protein affinity substance-immobilized carrier according to the present invention.
- the solution containing the protein RNA complex used in this case includes cell lysate (cell extract) containing the protein RNA complex, cell culture supernatant, body fluid such as plasma, serum, urine, saliva, breast milk, Or the solution which melt
- cell lysate a cell lysate obtained after containing 5 ⁇ 10 6 to 1 ⁇ 10 7 cells in 1 mL of a normal solution is used.
- a buffer solution containing NaCl or the like As a solvent (reaction solvent) of a solution containing a protein RNA complex when using a cell lysate, a buffer solution containing NaCl or the like can be mentioned, but it is usually used in this field in order to obtain small RNA efficiently.
- a buffer containing NaCl to which a surfactant as a cell lysing agent is further added is preferable.
- the buffer solution have a pH of about 5.0 to 10.0, preferably about pH 6.5 to 8.5, such as a phosphate buffer, Tris buffer, Good buffer, glycine buffer, and borate buffer. Etc. are preferred.
- the concentration of the buffering agent in the buffer is appropriately selected from the range of usually 10 to 500 ⁇ mM, preferably 10 to 300 ⁇ mM.
- the concentration of NaCl is usually 100 to 200 mM as the concentration in the buffer.
- examples of the surfactant to be added include poly (oxyethylene) nonylphenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.), Triton X-100 and the like, and poly (oxyethylene) nonylphenyl ether is preferable.
- the concentration may be any concentration that does not affect the protein affinity substance according to the present invention and has a cytolytic effect, and is usually 0.01 to 0.5% with respect to the total amount of the buffer solution, and 0.05 to 0.1%. Is preferred.
- a surfactant may be added as a solubilizer for cell secretory granules (exosomes).
- the surfactant include poly (oxyethylene) nonylphenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.), Triton X-100 and the like, and poly (oxyethylene) nonylphenyl ether is preferable.
- the added concentration may be any concentration that does not affect the protein affinity substance according to the present invention and has a cytolytic effect, and is usually 0.01 to 0.5% and 0.05 to 0.1% with respect to the total amount of the sample. Is preferred.
- the amount of the protein affinity substance-immobilized carrier according to the present invention used in the step (1) is usually 1 to 10 mg, preferably 1 to 5 mg per 1 mL of the solution containing the protein RNA complex.
- the amount of the protein affinity substance to be immobilized on the present invention is usually 0.1 to 100 ⁇ g, preferably 1 to 50 ⁇ g.
- step (2) After the reaction in the above step (1), if necessary, the carrier to which the protein RNA complex according to the present invention is bound is isolated from the reaction solution, and is further attached to the surface of the free protein RNA complex or cell. In order to remove the derived component, the obtained carrier is preferably washed with a washing solution.
- the reaction solution of step (1) is required, it is subjected to centrifugation and then the supernatant of the reaction solution is removed.
- the centrifugation is not particularly limited as long as it is usually carried out in this field. For example, it may be centrifuged at 1000 to 10000 ⁇ g for 10 to 100 seconds. Further, if the carrier is prepared using a magnetic material as a material, it is not necessary to centrifuge, and the carrier can be isolated by separating the carrier and the supernatant using a magnetic stand and removing the supernatant.
- washing solution used in the above washing examples include a buffer solution containing NaCl, and a buffer solution containing NaCl to which a surfactant as a cell lysing agent usually used in this field is further added is preferable.
- the buffer solution have a pH of about 5.0 to 10.0, preferably about pH 6.5 to 8.5, such as a phosphate buffer, Tris buffer, Good buffer, glycine buffer, and borate buffer. Etc. are preferred.
- the concentration of the buffering agent in the buffer is appropriately selected from the range of usually 10 to 500 ⁇ mM, preferably 10 to 300 ⁇ mM.
- the concentration of NaCl is usually 100 to 200 mM as the concentration in the buffer.
- the surfactant examples include poly (oxyethylene) nonylphenyl ether (manufactured by Wako Pure Chemical Industries, Ltd.), Triton X-100, and the like, and poly (oxyethylene) nonylphenyl ether is more preferable.
- the concentration may be any concentration that does not affect the protein affinity substance according to the present invention and has a cytolytic effect, and is usually 0.01 to 0.5% with respect to the total amount of the buffer solution, and 0.05 to 0.1%. It is.
- the amount of the cleaning solution used is usually 1 to 10 times the amount of the reaction solution in the above step (1).
- the washing operation By performing the washing operation as described above, it is possible to remove contaminants such as biological components attached to the sexually active substance-immobilized carrier according to the present invention, and in particular, Mg and Ca contained in the biological components. Divalent cations can be removed. As a result, the small RNA is not hydrolyzed by the divalent cation at a high temperature of 70 to 100 ° C., and the small RNA can be obtained with higher efficiency. Further, the above washing operation is preferably carried out repeatedly because the effect is enhanced by repeating the washing operation.
- step (3) After step (1) or after step (2), the carrier to which the protein RNA complex according to the present invention is bound is 10 to 100 ⁇ L, preferably 10 to 50 ⁇ L of water or pH of 3.0 to 8.0 per 1 mg of carrier.
- the buffer solution usually heat at 70 to 100 ° C. for 30 to 600 seconds. Thereafter, if necessary, the solution is centrifuged, and the supernatant of the solution is collected to obtain a solution containing the target small RNA.
- a solution can be used directly as a sample for a reverse transcription reaction or a PCR reaction.
- the water used in the above step (3) is not particularly limited as long as it is usually used in this field, but water from which nucleases such as ribonuclease and deoxyribonuclease have been removed is preferable.
- the buffer used in the above step (3) is hydrolyzed if the alkalinity is strong, so the pH is usually in the range of 3.0 to 8.0, and the RNA recovery rate is high and does not inhibit the subsequent reaction. Those having a pH of 4.5 to 7.0 are preferred.
- the type of buffer solution is not limited as long as it is in the above pH range, and specific examples include Good's buffer solution such as citrate buffer solution, MES (2-Morpholinoethanesulfonic acid), phosphate buffer solution, Tris buffer solution and the like. Of these, citrate buffer, Good's buffer, phosphate buffer, and the like are preferable, and citrate buffer, phosphate buffer, and the like are more preferable.
- the concentration of the buffer in the buffer is usually 5 to 100 mM, preferably 10 to 50 mM.
- small RNA can be obtained with higher efficiency than when water is used.
- the water or buffer used in the above step (3) may contain RNA or DNA that does not contain the base sequence of the target RNA.
- RNA dissolved in water or buffer include rRNA ( Ribosomal RNA), tRNA (transfer RNA), synthetic hairpin RNA, and the like. Among them, rRNA, synthetic hairpin RNA, and the like are preferable.
- DNA include those derived from salmon sperm and those derived from lambda phage. Among these, those derived from salmon sperm are preferred.
- RNA and DNA contained in the water or buffer varies depending on the RNA or DNA used, but is such an amount that the concentration in the solution is usually 0.1 to 10 ng / ⁇ L, preferably 0.1 to 1 ng / ⁇ L. .
- the solution used is preferably water rather than a buffer solution.
- the heating temperature of the water or buffer used in the above step (3) is usually 70 to 100 ° C, preferably 80 to 100 ° C, more preferably 80 to 90 ° C.
- the heating time may be set between 30 and 600 seconds depending on the heating temperature. Specifically, for example, 120 to 600 seconds at 70 ° C, 90 to 600 seconds at 80 ° C, and 30 to 90 ° C or more. ⁇ 600 seconds.
- the centrifugation in the above step (3) is not particularly limited as long as it is usually carried out in this field. For example, it may be centrifuged at 1000 to 10000 ⁇ g for 10 to 100 seconds.
- the method for obtaining small RNA of the present invention is more specifically performed as follows, for example, when Ago2 is used as the protein affinity substance according to the present invention. That is, for example, 1 to 20 mg, preferably 5 to 10 mg of the carrier of the present invention is added to 1 mL of plasma containing a complex of Ago2 and small RNA, and reacted at 2 to 10 ° C. for 2 to 24 hours. Furthermore, after the obtained reaction solution was centrifuged at 3000 to 5000 ⁇ g for 10 to 50 seconds to remove the supernatant, 0.05 to 0.1 w / v% poly (oxyethylene) nonylphenyl ether and 100 to 300 mM NaCl were contained.
- the carrier to which the complex is bound is washed several times with 1 to 5 mL of 10 to 100 mM Tris buffer (pH 7.0 to 8.0) per 1 mL of the reaction solution.
- the target small RNA is released by heating at 80 to 100 ° C. for 90 to 180 seconds using, for example, a citrate buffer of pH 4.5 to 6.0.
- the obtained small RNA is obtained by centrifuging the obtained solution at 3000 to 5000 ⁇ g for 10 to 50 seconds and fractionating the supernatant.
- Example 1 Acquisition of miRNA from human plasma by the heated immunoprecipitation elution method (target of acquisition: miR-92a)
- Anti-human Ago2 antibody immobilization carrier is Antibody Immobilization Kit IP (Wako Pure Chemical Industries, Ltd.) and 1 mg / mL anti-human Ago2 antibody (Wako Pure Chemical Industries, Ltd.) And manufactured as follows. That is, Antibody Immobilization Buffers (9.0 mL) and 1 mg / mL anti-human Ago2 antibody (1.0 mL) were added to and mixed with 200 mg of Antibody Immobilization Beads, and mixed by inverting at 37 ° C. for 4 hours.
- miRNA reverse transcription and quantitative PCR The reverse transcription reaction of miRNA was carried out as follows using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) and TaqMan MicroRNA Assay Kit (for hsa-miR-92a) (Applied Biosystems).
- Comparative Example 1 Acquisition of miRNA from human plasma by immunoprecipitation SDS / phenol / chloroform elution method (target: miR-92a) (1) Obtaining miRNA from human plasma by immunoprecipitation SDS / phenol / chloroform elution method Remove 50 ⁇ L of anti-human Ago2 antibody-immobilized carrier solution (equivalent to 2 mg of beads) after centrifugation (3000 ⁇ g, 30 seconds) and remove supernatant After washing once with 1 mL of the immunoprecipitation washing solution, 200 ⁇ L of a pretreated human plasma sample was added and mixed by inverting for 3 hours.
- anti-human Ago2 antibody-immobilized carrier solution equivalent to 2 mg of beads
- the upper layer was separated, 400 ⁇ L of chloroform was added thereto, mixed with a vortex mixer, and then centrifuged (20000 ⁇ g, 10 minutes).
- the upper layer was separated, added with 3 ⁇ L of Eta precipitated mate (manufactured by Nippon Gene Co., Ltd.), 40 ⁇ L of 3M sodium acetate and 1 mL of ethanol, suspended in a vortex mixer, and then centrifuged (20000 ⁇ g, 15 minutes).
- the obtained precipitate was washed with 1 mL of 70 v / v% ethanol, then air-dried at room temperature for 20 minutes, and the dried product dissolved in 50 ⁇ L of nuclease-free water was used as a measurement sample.
- Comparative Example 2 Acquisition of miRNA from human plasma by immunoprecipitation acidic elution method (target: miR-92a) (1) Obtaining miRNA from human plasma by immunoprecipitation acidic elution method Centrifugation (3000 xg, 30 seconds) of anti-human Ago2 antibody-immobilized carrier solution (equivalent to 2 mg of beads), remove supernatant, and immunoprecipitation washing solution After washing once with 1 mL, 200 ⁇ L of a pretreated human plasma sample was added and mixed by inverting for 3 hours in a refrigerator.
- miRNA equivalent to the method of phenol / chloroform extraction and ethanol precipitation after SDS elution is detected.
- elution of small RNA such as miRNA by the heat treatment is a simple method corresponding to high throughput without requiring operations such as nucleic acid extraction with phenol / chloroform and ethanol precipitation.
- Example 2 Effect on elution efficiency due to difference in heating temperature (Target: miR-92a) (1) Obtaining miRNA from human plasma by immunoprecipitation heat elution method Centrifugation (3000 xg, 30 seconds) of anti-human Ago2 antibody-immobilized carrier solution (equivalent to 2 mg of beads), remove the supernatant, and wash the immunoprecipitation After washing once with 1 mL, 200 ⁇ L of a pretreated human plasma sample was added and mixed by inverting (about 5 ° C.) for 3 hours. After centrifugation (3000 ⁇ g, 30 seconds), the supernatant was removed and washed 3 times with 1 mL of immunoprecipitation washing solution to obtain a precipitate (pellet).
- nuclease-free water (Nippon Gene Co., Ltd.) was added to the pellet and stirred at 1400 rpm using a thermomixer (Eppendorf).
- Eppendorf thermomixer
- Six types of solutions obtained in the same manner as described above were prepared and incubated at 50 ° C., 60 ° C., 70 ° C., 80 ° C., 90 ° C., and 100 ° C. for 5 minutes, respectively. After centrifugation (3000 ⁇ g, 30 seconds), supernatants were collected and used as measurement samples.
- the elution efficiency of miRNA from the carrier obtained by immunoprecipitation shows a higher elution rate as the temperature is higher, and miRNA is efficiently eluted at 70 ° C. or higher, and further at 80 ° C. or higher. It was found that it was eluted efficiently.
- Example 3 Effect on elution efficiency due to difference in heating temperature and heating time (Obtained object: miR-92a) (1) Obtaining miRNA from human plasma by immunoprecipitation heat elution method After centrifugation (3000 xg, 30 seconds) of 50 ⁇ L of anti-human Ago2 antibody-immobilized carrier solution (equivalent to 2 mg of beads), the supernatant is removed and immune Washing was performed once with 1 mL of the sedimentation washing solution. Next, 200 ⁇ L of a pretreated human plasma sample was added and mixed by inverting for 3 hours.
- Example 4 Effect on elution efficiency due to differences in buffer type and pH of heating solution (target: miR-92a)
- RNA can be eluted as efficiently as using water with a pH 3.0-8.0 buffer, or more efficiently than using water with a pH 4.5-7.0 buffer. It was found that RNA can be eluted well.
- Example 5 Effect on elution efficiency by additives added to heating solution (target: miR-92a)
- (1) Obtaining miRNA from human plasma by immunoprecipitation heat elution method Centrifugation (3000 xg, 30 seconds) of anti-human Ago2 antibody-immobilized carrier solution (equivalent to 2 mg of beads), remove the supernatant, and wash the immunoprecipitation Washed once with 1 mL. Next, 200 ⁇ L of a pretreated human plasma sample was added and mixed by inverting for 3 hours. Furthermore, after centrifugation (3000 ⁇ g, 30 seconds), the supernatant was removed, and the precipitate was washed three times with 1 mL of an immunoprecipitation washing solution to obtain a precipitate (pellet).
- nuclease-free water Nippon Gene Co., Ltd.
- E. coli ribosomal RNA rRNA, Wako Pure Chemical Industries, Ltd.
- Nuclease-free water added to a concentration of 1, 10 ng / ⁇ L
- Nuclease-free water added with salmon sperm DNA ssDNA, manufactured by Wako Pure Chemical Industries, Ltd.
- ssDNA salmon sperm DNA
- the E. coli ribosomal RNA was added to the solution used for the heating elution method at a final concentration of 0.1 to 10 ng / ⁇ L, or the salmon sperm DNA was added at a final concentration of 0.1 to 1 ng / ⁇ L. It was shown that the amount of miRNA recovered increases when added. That is, it was found that the RNA elution amount increased by adding an appropriate amount of nucleic acid to the heated solution. This was thought to be due to the effect of protecting miRNA from heat and the effect of preventing adsorption of miRNA to the container.
- Example 6 Immunoprecipitation heat elution method using various heating solutions (acquired: miR-16, miR-92a) (1) Obtaining miRNA from human plasma by immunoprecipitation heat elution method Centrifugation (3000 xg, 30 seconds) of anti-human Ago2 antibody-immobilized carrier solution (equivalent to 2 mg of beads), remove the supernatant, and wash the immunoprecipitation After washing once with 1 mL, 200 ⁇ L of a pretreated human plasma sample was added and mixed by inverting for 3 hours in a refrigerator.
- ssDNA manufactured by Wako Pure Chemical Industries, Ltd.
- diluted single-stranded miR-16 (5'-UAGCAGCACGUAAAUAUUGCGCG -3 ') as standard RNA
- a miR-16 calibration curve was created from the Ct value of each diluted standard RNA solution and the number of molecules. Using the calibration curve, the number of miR-16 molecules was calculated from the Ct values obtained above. The result is shown in FIG. 6 together with the result of miR-92a.
- the upper layer was separated, 400 ⁇ L of chloroform was added, mixed with a vortex mixer, and then centrifuged (20000 ⁇ g, 10 minutes).
- the upper layer was separated, added with Eta precipitated mate (manufactured by Nippon Gene Co., Ltd.) 3 ⁇ L, 3M sodium acetate 40 ⁇ L, and ethanol 1 mL, suspended in a vortex mixer, and then centrifuged (20000 ⁇ g, 15 minutes).
- the precipitate was washed with 1 mL of 70 v / v% ethanol, then air-dried at room temperature for 20 minutes, dissolved in 50 ⁇ L of nuclease-free water, and this solution was used as a measurement sample.
- the upper layer was separated, and 0.6 mL of isopropanol was added and mixed, followed by centrifugation (20000 ⁇ g, 10 minutes).
- the precipitate was taken out, washed with 1 mL of 70 v / v% ethanol, and then air-dried at room temperature for 20 minutes.
- the obtained dried product was dissolved in 50 ⁇ L of sterilized water, and the resulting solution was used as a measurement sample.
- nuclease-free water nuclease-free water with E. coli ribosomal RNA (final concentration 1 ng / ⁇ L)
- nuclease-free water with salmon sperm DNA final concentration 1 ng / ⁇ L
- solution of 10 mM citrate buffer (pH 5.5) is used for the heat treatment
- conventional immunoprecipitation methods that perform phenol / chloroform extraction and ethanol precipitation after SDS elution, and general purification of RNA It was shown that miR-16 in plasma can be obtained more efficiently than the AGPC method.
- any solution is equivalent to or equivalent to the conventional immunoprecipitation method in which phenol / chloroform extraction and ethanol precipitation are performed after SDS elution.
- the above recoveries were shown, and nuclease-free water with E. coli ribosomal RNA (final concentration 1 ng / ⁇ L), nuclease-free water with salmon sperm DNA (final concentration 1 ng / ⁇ L), and 10 mM citrate buffer (pH 5.5) were used.
- the immunoprecipitation heat elution method showed almost the same amount of recovery as the AGPC method.
- the immunoprecipitation heat elution method of the present invention can be carried out by a simple operation, it collects small RNA such as miRNA in plasma with an efficiency equal to or higher than that of the conventional method. It turned out to be an excellent way to do it.
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Abstract
Description
本願発明者らは、上記状況に鑑み鋭意研究を重ねた結果、免疫沈降法において、Argonauteファミリータンパク質-small RNA複合体を担持した担体を特定の溶液中で加熱することで複合体からsmall RNAを溶出することができ、溶出液を直接その後の反応系に用いることが可能となることを見出し、本発明を完成するに至った。即ち、本発明は、ハイスループットに適用することができ、簡便に効率よくsmall RNAを取得し得る方法の提供を課題とする。
更に、本発明の方法は、small RNAの取得効率に関しても、従来法と同等又はそれ以上の高い効率でsmall RNAを取得できる。従って、本発明の方法によれば、サンプル数が多い場合であっても短時間でsmall RNAの取得を可能とするため、多数のサンプルを扱う研究や診断等に適した方法である。
また、上記アプタマーの作製方法としては、米国特許第5,270,163号に記載の方法等が好ましい。
即ち、上記の如き本発明に係る担体1gに対して抗Ago2抗体等の本発明に係るタンパク質親和性物質0.1~10mg存在するようにして炭酸水素ナトリウム緩衝液中に添加し、30~40℃で、2~10時間反応させることにより、本発明に係るタンパク質親和性物質が粒子の表面上に固定化され、本発明に係るタンパク質親和性物質固定化担体が得られる。
[工程(1)]本発明に係るタンパク質親和性物質固定化担体と、本発明に係るタンパク質RNA複合体とを接触させて、当該担体にタンパク質RNA複合体を結合させ、
[工程(2)]要すれば、当該タンパク質RNA複合体が結合した担体を洗浄し、
[工程(3)]当該タンパク質RNA複合体が結合した担体を、水又はpHが3.0~8.0の緩衝液中で70~100℃で加熱し、small RNAを遊離させることによりなされる。
まず、タンパク質RNA複合体を含む溶液と本発明に係るタンパク質親和性物質固定化担体とを混合し、2~37℃、好ましくは2~10℃で1~30時間、好ましくは2~10時間、より好ましくは2~5時間反応させ、本発明に係るタンパク質親和性物質固定化担体に、本発明に係るタンパク質RNA複合体を結合させる。
上記工程(1)の反応の後、要すれば、本発明に係るタンパク質RNA複合体が結合した担体を、反応溶液から単離し、更に、担体表面に付着した、遊離のタンパク質RNA複合体や細胞由来成分を取り除くため、得られた担体を洗浄液で洗浄するのが好ましい。
工程(1)の後または工程(2)の後、本発明に係るタンパク質RNA複合体が結合した担体を、担体1mgに対して10~100μL、好ましくは10~50μLの水又はpHが3.0~8.0の緩衝液中で、通常70~100℃で30~600秒加熱する。その後、要すれば当該溶液を遠心分離に付した後、該溶液の上清を分取することにより、目的のsmall RNAを含有する溶液を得る事ができる。このような溶液は、逆転写反応やPCR反応の試料として直接用いることができる。
(1)抗ヒトAgo2抗体固定化担体の作製
抗ヒトAgo2抗体固定化担体はAntibody Immobilization Kit IP(和光純薬工業(株)製)と1mg/mL抗ヒトAgo2抗体(和光純薬工業(株)製)を用いて下記のように作製した。
即ち、Antibody Immobilization Beads 200mgにAntibody Immobilization Buffer 9.0mLと1mg/mL抗ヒトAgo2抗体1.0mLを添加混合し、37℃で4時間転倒混和した。遠心分離(3000×g、5分間)後、上清をピペットで除き、Washing Buffer(Neutral) 10mLで洗浄を3回行った。その後、Blocking Buffer 10mLを入れ、室温で1時間転倒混和した。遠心分離(3000×g、5分間)後、上清をピペットで除き、Washing Buffer(Neutral) 10mLで洗浄を5回行い、Storage Buffer10mLで洗浄を1回行った後、Storage Buffer5.0mLでビーズを懸濁し、抗ヒトAgo2抗体固定化担体溶液(以下、このようにして得た溶液を、抗ヒトAgo2抗体固定化担体溶液と記載する)を得た。
ヒトプール血漿(コージンバイオ(株)製)1mLを遠心分離(20000×g、15分間)した後、上清を新しいチューブに移し、ポリ(オキシエチレン)ノニルフェニルエーテル(和光純薬工業(株)製)を終濃度0.1 w/v %となるように添加したものを、ヒト血漿サンプルとして用いた(以下、このように前処理したヒト血漿サンプルを前処理済みヒト血漿サンプルと記載する)。
上記(1)で得た抗ヒトAgo2抗体固定化担体溶液50μL(ビーズ2mg相当量含量)を遠心分離(3000×g、30秒間)後上清を除き、0.05w/v%ポリ(オキシエチレン)ノニルフェニルエーテル(和光純薬工業(株)製)及び200mM塩化ナトリウム含有20mMトリス塩酸水溶液(pH7.4、以下、免疫沈降洗浄液と略記する)1mLで1回洗浄を行った後、前記処理済みヒト血漿サンプル200μLを添加し、冷蔵で3時間転倒混和した。遠心分離(3000×g、30秒間)後上清を除き、免疫沈降洗浄液1mLで3回洗浄した。その後ペレットにヌクレアーゼフリー水(ニッポンジーン(株)製)50μLを添加し、サーモミキサー(エッペンドルフ社製)を用いて1400rpmで攪拌しながら95℃で5分間インキュベーションした。遠心分離(3000×g、30秒間)の後、上清を分取し、得られたものを測定用試料とした。
miRNAの逆転写反応はTaqMan MicroRNA Reverse Transcription Kit(アプライドバイオシステムズ社製)およびTaqMan MicroRNA Assay Kit(hsa-miR-92a用)(アプライドバイオシステムズ社製)を用いて下記のように行った。
即ち、上記方法で得られた測定用試料5μLに、100mM dNTP mix 0.15μL、10×RT Buffer 1.5μL、20U/μL RNase Inhibitor 0.19μL、50U/μL Multiscribe RT enzyme 1μL 、5×RT primer( hsa-miR-92a用)3μL、精製水4.16μLを添加しtotal 15μLの反応液を調製した。各反応液を 16℃で30分間、42℃で30分間インキュベーションした後、85℃で5分間インキュベーションし、逆転写反応産物を得た。
得られた逆転写反応産物1μLに、TaqMan 2×PCR Master Mix (アプライドバイオシステムズ社製)7.5μL、20×TaqMan Assay Mix( hsa-miR-92a用)0.75μL、精製水5.75μLを添加し、total 15μLの反応液を調製し、ABI7500 Fast Real-Time PCR System(アプライドバイオシステムズ社製)を用いて定量PCR検出を行い、各Ct値を求めた。
一方、1本鎖合成miR-92a(5'- UAUUGCACUUGUCCCGGCCUGU -3')を標準RNAとし、精製水で101~105copies/μLに10倍希釈した希釈溶液を調製し、各希釈溶液1μLを上記と同様の方法で逆転写反応させ定量PCR検出を行った。得られた各希釈標準RNA溶液のCt値とその分子数から、miR-92aの検量線を作成した。該検量線を用いて、上記で得られた各Ct値よりmiR-92aの分子数を算出した。その結果を、図1に示す。
(1) 免疫沈降SDS・フェノール/クロロホルム溶出法によるヒト血漿からのmiRNA取得
抗ヒトAgo2抗体固定化担体溶液50μL(ビーズ2mg相当量)を遠心分離(3000×g、30秒間)後上清を除き、免疫沈降洗浄液1mLで1回洗浄を行った後、前処理済みヒト血漿サンプル200μLを添加し、冷蔵で3時間転倒混和した。遠心分離(3000×g、30秒間)後上清を除き、免疫沈降洗浄液1mLで3回洗浄し、沈殿物(ペレット)を得た。その後、該ペレットに0.5w/v% SDS溶液50μLを添加し、担体に結合したタンパク質を溶出した。更に、溶出液に滅菌水350μL、フェノール:クロロホルム:イソアミルアルコール(25:24:1)400μLを添加し、ボルテックスミキサーで混合した後、遠心分離(20000×g、10分間)した。上層を分取し、これにクロロホルム400μLを添加し、ボルテックスミキサーで混合後、遠心分離(20000×g、10分間)した。その上層を分取し、エタ沈メイト(ニッポンジーン(株)製) 3μL、3M酢酸ナトリウム 40μL、エタノール 1mLを加え、ボルテックスミキサーで懸濁後、遠心分離(20000×g、15分間)した。得られた沈殿物を70 v/v %エタノール1mLで洗浄後、室温で20分間風乾し、その乾燥物をヌクレアーゼフリー水50μLに溶解したものを、測定用試料とした。
得られた測定用試料を用いて、実施例1(4)記載の方法と同様に、逆転写反応および定量PCR検出を行い、得られたCt値からmiR-92aの分子数を算出した。その結果を、図1に実施例1の結果と併せて示す。
(1)免疫沈降酸性溶出法によるヒト血漿からのmiRNA取得
抗ヒトAgo2抗体固定化担体溶液50μL(ビーズ2mg相当量)を遠心分離(3000×g、30秒間)後上清を除き、免疫沈降洗浄液1mLで1回洗浄を行った後、前処理済みヒト血漿サンプル200μLを添加し、冷蔵で3時間転倒混和した。遠心分離(3000×g、30秒間)後上清を除き、免疫沈降洗浄液1mLで3回洗浄し、沈殿物(ペレット)を得た。その後、該ペレットに0.1Mグリシン-塩酸緩衝液(pH2.5)46.5μLを添加し、ボルテックスミキサーで混合後、遠心分離(3000×g、30秒間)を行い、上清を分取し、1M CAPS(pH11.0) 3.5μLを添加して中和した溶液を測定用試料とした。
得られた測定用試料を用いて、実施例1(4)記載の方法と同様に、逆転写反応および定量PCR検出を行い、得られたCt値からmiR-92aの分子数を算出した。その結果を、図1に実施例1の結果と併せて示す。
(1)免疫沈降加熱溶出法によるヒト血漿からのmiRNA取得
抗ヒトAgo2抗体固定化担体溶液50μL(ビーズ2mg相当量)を遠心分離(3000×g、30秒間)後上清を除き、免疫沈降洗浄液1mLで1回洗浄を行った後、前処理済みヒト血漿サンプル200μLを添加し、冷蔵(約5℃)で3時間転倒混和した。遠心分離(3000×g、30秒間)後上清を除き、免疫沈降洗浄液1mLで3回洗浄し、沈殿物(ペレット)を得た。その後、該ペレットにヌクレアーゼフリー水(ニッポンジーン(株)製)50μLを添加し、サーモミキサー(エッペンドルフ社製)を用いて1400rpmで攪拌した。上記操作と同様にして得た溶液を6種類準備し、それぞれ、50℃、60℃、70℃、80℃、90℃、100℃で5分間インキュベーションした。遠心分離(3000×g、30秒間)の後、上清を分取し、それらを測定用試料とした。
得られた各測定用試料を用いて、実施例1(4)記載の方法と同様に、逆転写反応および定量PCR検出を行い、得られたCt値からmiR-92aの分子数を算出した。その結果を、図2に示す。
(1)免疫沈降加熱溶出法によるヒト血漿からのmiRNA取得
抗ヒトAgo2抗体固定化担体溶液50μL(ビーズ2mg相当量)を遠心分離(3000×g、30秒間)した後、上清を除き、免疫沈降洗浄液 1mLで1回洗浄を行った。次いで、前処理済みヒト血漿サンプル200μLを添加し、冷蔵で3時間転倒混和した。更に、遠心分離(3000×g、30秒間)後上清を除き、免疫沈降洗浄液1mLで3回洗浄し、沈殿物(ペレット)を得た。該ペレットにヌクレアーゼフリー水(ニッポンジーン(株)製)50μLを添加した。上記操作と同様にして得た溶液を24種類準備し、サーモミキサー(エッペンドルフ社製)を用いて1400rpmで攪拌しながら、それぞれ70℃、80℃、90℃、100℃で10、30、60、90、120、180秒間インキュベーションした。最後に、遠心分離(3000×g、30秒間)の後、上清を分取し、それらを免疫沈降溶出液とした。
得られた測定用試料を用いて、実施例1(4)記載の方法と同様に、逆転写反応および定量PCR検出を行い、得られたCt値からmiR-92aの分子数を算出した。その結果を、図3に示す。
(1)免疫沈降加熱溶出法によるヒト血漿からのmiRNA取得
抗ヒトAgo2抗体固定化担体溶液50μL(ビーズ2mg相当量)を遠心分離(3000×g、30秒間)し、その後上清を除き、免疫沈降洗浄液1mLで1回洗浄を行った。次いで、前処理済みヒト血漿サンプル200μLを添加し、冷蔵で3時間転倒混和した。更に、遠心分離(3000×g、30秒間)後上清を除き、免疫沈降洗浄液1mLで3回洗浄した後、沈殿物(ペレット)を得た。上記操作と同様にして得た溶液を11種類を準備し、ペレットそれぞれに、ヌクレアーゼフリー水(ニッポンジーン(株)製)、10mMクエン酸緩衝液(pH3.0)、10mMクエン酸緩衝液(pH4.0)、10mMクエン酸緩衝液(pH4.5)、10mMクエン酸緩衝液(pH5.0)、10mMクエン酸緩衝液(pH5.5)、10mMクエン酸緩衝液(pH6.0)、10mM MES緩衝液(pH6.5)、10mMリン酸緩衝液(pH7.0)、10mMリン酸緩衝液(pH8.0)又は10mMトリス塩酸緩衝液(pH8.0)各50μLを添加し、サーモミキサー(エッペンドルフ社製)を用いて1400rpmで攪拌しながら90℃で90秒間インキュベーションした。最後に、遠心分離(3000×g、30秒間)の後、上清を分取し、それらを測定用試料とした。
得られた各測定用試料を用いて、実施例1(4)記載の方法と同様に、逆転写反応および定量PCR検出を行い、得られたCt値からmiR-92aの分子数を算出した。その結果を、図4に示す。
(1)免疫沈降加熱溶出法によるヒト血漿からのmiRNA取得
抗ヒトAgo2抗体固定化担体溶液50μL(ビーズ2mg相当量)を遠心分離(3000×g、30秒間)後上清を除き、免疫沈降洗浄液1mLで1回洗浄を行った。次いで、前処理済みヒト血漿サンプル200μLを添加し、冷蔵で3時間転倒混和した。更に、遠心分離(3000×g、30秒間)後上清を除き、免疫沈降洗浄液1mLで3回洗浄し、沈殿物(ペレット)を得た。上記操作と同様にして得た溶液を7種類を準備し、ペレットそれぞれに、ヌクレアーゼフリー水(ニッポンジーン(株)製)、大腸菌リボゾーマルRNA(rRNA、和光純薬工業(株)製)を終濃度0.1、1、10ng/μLとなるように添加したヌクレアーゼフリー水、サケ精子DNA(ssDNA、和光純薬工業(株)製)を終濃度0.1、1、10ng/μLとなるように添加したヌクレアーゼフリー水各50μLを添加し、サーモミキサー(エッペンドルフ社製)を用いて1400rpmで攪拌しながら90℃で90秒間インキュベーションした。遠心分離(3000×g、30秒間)の後、上清を分取し、測定用試料とした。
得られた各測定用試料を用いて、実施例1(4)記載の方法と同様に、逆転写反応および定量PCR検出を行い、得られたCt値からmiR-92aの分子数を算出した。その結果を、図5に示す。
(1)免疫沈降加熱溶出法によるヒト血漿からのmiRNA取得
抗ヒトAgo2抗体固定化担体溶液50μL(ビーズ2mg相当量)を遠心分離(3000×g、30秒間)後上清を除き、免疫沈降洗浄液1mLで1回洗浄を行った後、前処理済みヒト血漿サンプル200μLを添加し、冷蔵で3時間転倒混和した。遠心分離(3000×g、30秒間)後上清を除き、免疫沈降洗浄液1mLで3回洗浄し、沈殿物(ペレット)を得た。上記操作と同様にして得た溶液4種類を準備し、各ペレットにヌクレアーゼフリー水(ニッポンジーン(株)製)、大腸菌リボゾーマルRNA(rRNA、和光純薬工業(株)製)を終濃度1ng/μLとなるように添加したヌクレアーゼフリー水、サケ精子DNA(ssDNA、和光純薬工業(株)製)を終濃度1ng/μLとなるように添加したヌクレアーゼフリー水、10mMクエン酸緩衝液(pH5.5)50μLを添加し、サーモミキサー(エッペンドルフ社製)を用いて1400rpmで攪拌しながら90℃で90秒間インキュベーションした。遠心分離(3000×g、30秒間)の後、上清を分取し、測定用試料とした。
miR-92a量の測定に関しては、得られた各測定用試料を用いて、実施例1(4)記載の方法と同様に、逆転写反応および定量PCR検出を行い、得られたCt値からmiR-92aの分子数を算出した。
即ち、得られた各測定用試料5μLに、100mM dNTP mix 0.15μL、10×RT Buffer 1.5μL、20U/μL RNase Inhibitor 0.19μL、50U/μL Multiscribe RT enzyme 1μL 、5×RT primer(hsa-miR-16用)3μL、精製水4.16μLをそれぞれ添加しtotal 15μLの反応液を調製した。各反応液を16℃で30分間、42℃で30分間インキュベーションした後、85℃で5分間インキュベーションし、逆転写反応産物を得た。
次いで、各逆転写反応産物1μLに、TaqMan 2×PCR Master Mix (アプライドバイオシステムズ社製)7.5μL、20×TaqMan Assay Mix(hsa-miR-16用)0.75μL、精製水5.75μLを添加し、total 15μLの反応液を調製し、ABI7500 Fast Real-Time PCR System(アプライドバイオシステムズ社製)を用いて定量PCR検出を行い、各Ct値を求めた。
一方、1本鎖合成miR-16(5'- UAGCAGCACGUAAAUAUUGGCG -3')を標準RNAとして、精製水で101~105copies/μLに10倍希釈した希釈溶液を調製し、その各希釈溶液1μLを上記と同様の方法で逆転写反応させて定量PCR検出を行った。得られた各希釈標準RNA溶液のCt値とその分子数から、miR-16の検量線を作成した。該検量線を用いて、上記で得られた各Ct値よりmiR-16の分子数を算出した。その結果を、miR-92aの結果と併せて図6に示す。
(1)免疫沈降従来法SDS・フェノール/クロロホルム溶出によるmiRNAの精製
抗ヒトAgo2抗体固定化担体溶液50μL(ビーズ2mg相当量)を遠心分離(3000×g、30秒間)後上清を除き、免疫沈降洗浄液1mLで1回洗浄を行った後、前処理済みヒト血漿サンプル200μLを添加し、冷蔵で3時間転倒混和した。遠心分離(3000×g、30秒間)後上清を除き、免疫沈降洗浄液1mLで3回洗浄し、沈殿物(ペレット)を得た。その後、該ペレットに2w/v% SDS溶液50μLを添加し、担体に結合したタンパク質を溶出した。溶出液に滅菌水350μL、フェノール:クロロホルム:イソアミルアルコール(25:24:1)400μLを添加し、ボルテックスミキサーで混合後、遠心分離(20000×g、10分間)した。上層を分取し、クロロホルム400μLを添加し、ボルテックスミキサーで混合後、遠心分離(20000×g、10分間)した。上層を分取し、エタ沈メイト(ニッポンジーン(株)製) 3μL、3M酢酸ナトリウム 40μL、エタノール 1mLを加え、ボルテックスミキサーで懸濁後、遠心分離(20000×g、15分間)した。沈殿を70 v/v %エタノール1mLで洗浄後、室温で20分間風乾し、ヌクレアーゼフリー水50μLに溶解し、該溶液を測定用試料とした。
得られた測定用試料を用いて、実施例6(2)記載の方法と同様に、逆転写反応および定量PCR検出を行い、miR-16及びmiR-92aの分子数を算出した。その結果を、図6に実施例6の結果と併せて示す。
(1)AGPC法によるmiRNA精製
前処理済みヒト血漿サンプル200μLにISOGEN-LS(ニッポンジーン(株)製)600μLとグリコーゲン(和光純薬工業(株)製)2μLを添加し、ボルテックスミキサーにより2分間混合した後、クロロホルム160μLを添加し、ボルテックスミキサーで10分間混合した。更に、遠心分離(20000×g、10分間)後、上層を分取し、イソプロパノール0.6mLを添加混合し遠心分離(20000×g、10分間)した。次いで、沈殿物を取り出し、70 v/v %エタノール1mLで洗浄後、室温で20分間風乾した。得られた乾燥物を、滅菌水50μLに溶解し、得られた溶液を測定用試料とした。
得られた各測定用試料を用いて、実施例6(2)記載の方法と同様に、逆転写反応および定量PCR検出を行い、miR-16及びmiR-92aの分子数を算出した。その結果を、図6に実施例6及び比較例3の結果と併せて示す。
また、miR-92aに関しても、本発明の免疫沈降加熱溶出法によれば、何れの溶液を用いても、SDS溶出の後フェノール/クロロホルム抽出とエタノール沈殿を行う従来の免疫沈降法と同等若しくはそれ以上の回収量を示し、大腸菌リボゾーマルRNA添加ヌクレアーゼフリー水(終濃度1ng/μL)、サケ精子DNA添加ヌクレアーゼフリー水(終濃度1ng/μL)、10mMクエン酸緩衝液(pH5.5)を用いた免疫沈降加熱溶出法はAGPC法とほぼ同等の回収量を示した。
従って、この結果から、本発明の免疫沈降加熱溶出法は、簡便な操作で実施できるにもかかわらず、従来法と比較して同等若しくはそれ以上の効率で血漿中のmiRNA等のsmall RNAを回収できる優れた方法であることが判った。
Claims (7)
- small RNA結合タンパク質に対して親和性を有する物質をその表面に固定化した担体と、small RNA結合タンパク質とsmall RNAの複合体(タンパク質RNA複合体)とを接触させて、当該担体にタンパク質RNA複合体を結合させ、
該タンパク質RNA複合体が結合した担体を、水又はpHが3.0~8.0の緩衝液中で70~100℃に加熱してsmall RNAを遊離させて取得することを特徴とする、small RNAの取得方法。 - 前記担体に前記タンパク質RNA複合体を結合させた後、加熱する前に、得られたタンパク質RNA複合体が結合した担体を洗浄する、請求項1記載の取得方法。
- 担体を緩衝液中で加熱する、請求項1又は2記載の取得方法。
- 緩衝液のpHが4.5~7.0である、請求項1~3の何れかに記載の取得方法。
- 緩衝液が、クエン酸緩衝液、Good's緩衝液又はリン酸緩衝液である、請求項1~4の何れかに記載の取得方法。
- 加熱が、30~600秒間の加熱である、請求項1~5の何れかに記載の取得方法。
- 水又は緩衝液が、対象のRNAの塩基配列を含まないRNA、又は、DNAを含有するものである、請求項1~6の何れかに記載の取得方法。
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CN201180045656XA CN103119163A (zh) | 2010-09-24 | 2011-09-20 | 小rna的获得方法 |
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WO2016088689A1 (ja) * | 2014-12-05 | 2016-06-09 | 和光純薬工業株式会社 | Timタンパク質結合担体、当該担体を用いた細胞外膜小胞及びウイルスの取得方法、除去方法、検出方法並びに当該担体を含むキット |
JP2017501689A (ja) * | 2013-11-27 | 2017-01-19 | シグマ−アルドリッチ・カンパニー・リミテッド・ライアビリティ・カンパニーSigma−Aldrich Co., LLC | 生物学的流体からのマイクロrna単離 |
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WO2021132245A1 (ja) | 2019-12-27 | 2021-07-01 | 富士フイルム和光純薬株式会社 | 転移性去勢抵抗性前立腺癌の診断を補助する方法 |
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