WO2007097119A1 - SUPPORT FOR CAPTURE OF mRNA, AND mRNA PURIFICATION METHOD - Google Patents

Abstract

Disclosed is a carrier for use in the capture of mRNA, which comprises an insoluble support and a polymeric substance provided on the surface of the support. The polymeric substance comprises: a first unit having a group derived from a phosphate ester that constitutes the hydrophilic moiety of a phospholipid; and a second unit having a carboxylic acid-derived group in which an electron-withdrawing substituent is bound to a carbonyl group. A DNA chain containing an oligo-dT sequence is bound to all or some of the carboxylic acid-derived groups at the 5'-terminus of the oligo-dT sequence.

Description

 Specification

 mRN A capture carrier and purification method of mRNA

 Technical field

 [0001] The present invention relates to a carrier for capturing mRNA and a method for purifying mRNA that purifies mRNA from a sample solution containing mRNA prepared from cells or the like.

 Background art

 [0002] As a method of observing responsiveness to stress on cells, it is a common practice to identify the amount of protein in the cell and to observe the gene expression status in the cell. The gene expression status is identified by the amount of mRNA of a specific gene. For gene detection, generally, total RNA is purified, the gene is amplified from the purified total RNA by RT-PCR method, and then mRNA is detected by electrophoresis or microphone array method. A method is used to check the gene expression level. According to the microarray method, the expression levels of multiple genes can be observed simultaneously. However, to see the gene expression level more accurately, it is better to purify only mRNA and apply the purified mRNA to RT-PCR.

 For purification of mRNA, the method using hybridization between the poly A-til moiety of the mRNA strand and the dT sequence is the mainstream. As a method for purifying mRNA, a method using magnetic beads is specified in 2002. -504348 (Patent Document 1) and Special Table 2002-535012 (Patent Document 2). In this method, the oligo d T chain is immobilized on the magnetic beads, the magnetic beads are put into a sample solution containing mRNA, the mRNA is captured on the beads, the beads are collected by a magnet, the sample solution is discarded, Move away the magnet, add cleaning solution, disperse, repeat the process of collecting the beads again with the magnet, and remove the mRNA.

[0003] In the purification of mRNA using magnetic beads, the problem was that the magnetic beads themselves were expensive. In addition, it is necessary to use a magnetic material for handling, and there is a problem that the operation is troublesome. In addition, conventional mR The NA purification method is poor in mRNA capture efficiency, and gene expression by RT-PCR requires amplification by multiple thermal cycles for gene expression. There was a problem of requiring. Patent Document 1: Japanese Translation of Special Publication 2002-504348

 Patent Document 2: Special Table 2002- 5350 12

 Disclosure of the invention

 Problems to be solved by the invention

 [0004] An object of the present invention is to provide a carrier for capturing mRNA and a method for purifying mRNA that are simple in operation and capable of purifying mRNA with high efficiency.

 Means for solving the problem

 [0005] The present inventors have found that mRNA can be purified easily and efficiently by using an insoluble carrier having a predetermined polymer substance introduced on the surface, and the present invention has been completed.

 The present invention

 (1) A first unit having a group derived from a phosphate ester constituting the hydrophilic part of the phospholipid and a carboxylic acid derivative group formed by bonding an electron-withdrawing substituent to a carbonyl group on the surface of the insoluble carrier. And a DNA substance containing an oligo dT sequence in all or part of the carboxylic acid derivative group is bonded to the 5 ′ end of the oligo dT sequence. A carrier for capturing mRNA

(2) The carrier for capturing mRNA according to (1), wherein the oligo dT sequence has 5 to 50 bases,

 (3) The form of the insoluble carrier is a container or a plate having a plurality of wells (1) or (2)

 (4) The form of the insoluble carrier is a porous body, (1) or (2) the carrier for capturing mRNA according to (2),

(5) The material for the insoluble carrier is plastic (1) to (4) any of the carrier for capturing mRNA. (6) Using the mRNA capture carrier according to any one of (1) to (5),

(a) supplying a sample solution containing mRNA to the carrier for capturing mRNA

(b) The process of hybridizing mRNA and oligo dT sequence-containing DNA,

 (c) supplying cleaning solution and cleaning,

 A method for purifying mRN A, comprising:

 (7) Further, (d) a step of eluting the hybridized mRNA, the method for purifying mRNA according to (6),

 (8) The method for purifying mRNA according to (7), wherein the sample solution containing mRNA is a solution in which cells are solubilized.

 It is.

 The invention's effect

 [0006] According to the present invention, mRNA can be purified with simple operation and high efficiency. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, embodiments of the present invention will be described in detail.

 [0008] The carrier for capturing mRNA according to the present embodiment has a first unit having a group derived from a phosphate ester constituting a hydrophilic part of a phospholipid on the surface of an insoluble carrier and an electron-withdrawing substituent is a carbonyl group. A DNA substance having an oligod T sequence in all or part of the carboxylic acid derivative group. 'It is characterized by bonding to the end.

[0009] Examples of insoluble carriers include polymers obtained from nature and derivatives thereof, and synthetic polymers and derivatives thereof. The former includes polysaccharides and their derivatives, or inorganic polymers such as glass and silica gel. The latter includes vinyl polymers such as polystyrene, polyethylene, and polypropylene. [0010] As the material of the insoluble carrier, plastic can be used.

 When plastic is used as the material for the insoluble carrier, flexibility in changing the shape and size is secured, and it is preferable from the viewpoint that it can be provided at a lower cost than that of glass. As such a plastic material, a thermoplastic resin can be used from the viewpoint of easy surface treatment and mass productivity.

 [001 1] As the thermoplastic resin, for example, linear polyolefin such as polyethylene and polypropylene, cyclic polyolefin, fluorine-containing resin and the like can be used.

 [001 2] The form of the insoluble carrier can be a container or a plate having a plurality of wells.

 As the container, a sampling tube or a tube for PCR can be used. If the capacity is 1 mL or less, it can be suitably used.

 In addition, plates having a plurality of wells can be suitably used.

 [001 3] The form of the insoluble carrier can be a porous body.

 The porous body is not particularly limited as long as it can form a porous body, including a sintered body and a non-woven fabric, but a sintered body is preferable considering that a large number of pore surfaces can be taken. A plastic porous body can be easily obtained by filling polyethylene and polypropylene fine particles into a mold and applying heat and pressure. Since the plastic porous body is heated in the manufacturing process, RNase can be deactivated. This is advantageous for applications dealing with R N A. In addition, it becomes possible to easily remove RNase, which is a problem by decomposing RNase, and this is advantageous in production management of the carrier for capturing mRNA of the present invention.

 [0014] The size of the pores requires an average surface diameter of 1 m to 50 because it is necessary to increase the surface area in the porous body and to allow the sample solution and the like to easily spread throughout the carrier. m is preferred.

[001 5] On the surface of the insoluble carrier of the present embodiment, phosphorus constituting the hydrophilic part of the phospholipid is formed. In the carboxylic acid derivative group portion of the polymer substance, the polymer includes a first unit having a group derived from an acid ester and a second unit having a carboxylic acid derivative group in which an electron-withdrawing substituent is bonded to a carbonyl group Oligo d T strand or DNA strand containing oligo d T strand is bound.

 [001 6] A first unit having a group derived from a phosphate ester constituting the hydrophilic part of the phospholipid and a carboxylic acid-derived group having a carboxylic acid-derived group formed by bonding an electron-withdrawing substituent to a carbonyl group. The polymer substance containing two units is a polymer having both the property of suppressing nonspecific adsorption of DNA strands and RNA strands and the property of immobilizing DNA strands. In particular, the phosphorylcholine group contained in the first unit plays a role in suppressing nonspecific adsorption of the truncated RNA fragment, and the carboxylic acid derivative group contained in the second unit plays a role in chemically immobilizing the dT chain. Fulfill. That is, the oligo d T chain is covalently bonded at the site of the carboxylic acid derivative group contained in the second unit.

 [001 7] The first unit is, for example, a (meth) acryloyloxyalkylphosphorylcholine group such as a 2-methacryloyloxychetylphosphorylcholine group, a 6-methacryloyloxyhexylphosphorylcholine group;

 (Meth) acryloyloxyalkoxyalkylphosphorylcholine groups such as 2_methacryloyloxychetoxyl phosphorylcholine group and 10_methacryloyloxyethoxynonylphosphorylcholine group;

 Alkenylphosphorylcholine groups such as allylphosphorylcholine group, butenylphosphorylcholine group, hexenylphosphorylcholine group, octenylphosphorylcholine group, and decenylphosphorylcholine group;

 And a phosphorylcholine group is included in these groups.

 [0018] Of these groups, 2-methacryloyloxychetyl phosphorylcholine is preferable. By adopting a structure in which the first unit has 2_methacryloyloxychetyl phosphorylcholine, nonspecific adsorption other than mRNA contained in the sample solution can be reliably suppressed.

[0019] Carboxylic acid derivatives are activated carboxylic acid carboxyl groups. Yes, a carboxylic acid having a leaving group via c = o. Specifically, a carboxylic acid derivative is a compound in which a nucleophilic reaction is activated by bonding a group having a higher electron withdrawing property than an alkoxyl group to a carbonyl group. The carboxylic acid derivative group is a compound having reactivity with an amino group, a thiol group, a hydroxyl group and the like.

 [0020] More specifically, as the activated carboxylic acid derivative, carboxyl groups such as carboxylic acid such as acrylic acid, methacrylic acid, crotonic acid, maleic acid, fumaric acid, acid anhydrides, acid halides, Examples include activated esters and compounds converted to activated amides. Carboxylic acid-derived groups are activated groups derived from such compounds, for example, active ester groups such as p-nitrophenyl groups or N_hydroxysuccinimide groups;

 — Halogens such as C I,-F;

 And the like.

 [0021] The carboxylic acid-derived group may be a group represented by the following formula (1).

 [0022] [Chemical 1] One C One A

 II (1)

 o

 (However, in the above formula (1), A is a leaving group excluding a hydroxyl group.)

[0023] The monovalent group represented by the above formula (1) can be, for example, any group selected from the following formula (p) or formula (q).

 Further, the following formula (t) or the following formula (V) may be used. In the above (1), A can be a substituent containing a nitrogen atom. A can be a halogen atom.

[0024] [Chemical 2]

C— O— CR ¹

― C101 NR2

 II (q)

 O

[0025] (However, in the above formulas (p) and (q), and, respectively, each independently represents a monovalent organic group, which may be linear, branched, or cyclic. In the above formula (p), R ¹ may be a divalent group that forms a ring together with C. In the above formula (q), R ² may be a ring together with N. It may be a divalent group that forms.

 [0026] Examples of the group represented by the formula (p) include groups represented by the following formulas (r), (s), and (w). Examples of the group represented by the above formula (q) include a group represented by the following formula (u).

 [0027] The group represented by the above formula (1) is, for example, a group derived from an acid anhydride represented by the following formula (r), formula (s), etc .;

 A group derived from an acid halide represented by the following formula (t);

A group derived from an active ester represented by the following formula (u) or (w); or a group derived from an activated amide represented by the following formula (V). [Chemical 3]

[0028] Of the carboxylic acid-derived groups, an active ester group is preferably used because of its excellent reactivity under mild conditions. The mild conditions include, for example, neutral or alkaline conditions, specifically pH 7.0 or more and 10.0 or less, more specifically pH 7.6 or more and 9.0 or less, and more specifically Specifically, it can be set to pH 8.0.

 [0029] Further, the definition of "active ester group" as defined in this specification is not strictly defined. However, as a conventional technical expression, acidity is present on the alcohol side of the ester group. An ester group that has a high electron-withdrawing group and activates a nucleophilic reaction, that is, an ester group with high reaction activity, is used in various chemical synthesis fields such as polymer chemistry and peptide synthesis. It is the one that is commonly used. In the field of peptide synthesis, as described in Nobuo Izumiya, Tetsuo Kato, Toshihiko Aoyagi, Noriwaki Waki, “Basics and Experiments of Peptide Synthesis”, published in 1958, Maruzen, The active ester method is used as one of the methods for activating the C-terminus of amino acids or peptides.

[0030] Actually, the ester group has an electron-withdrawing group on the alcohol side of the ester group and is activated more than the alkyl ester. The active ester group has reactivity with groups such as an amino group, a thiol group, and a hydroxyl group. More specifically, phenol esters, thiophenol esters, N-hydroxy Amamine esters, cyanomethyl esters, esters of heterocyclic hydroxy compounds, and the like are known as active ester groups having much higher activity than alkyl esters and the like.

 [0031] Here, the case where the carboxylic acid derivative group in the polymer substance is an active ester group will be described as an example. Examples of the active ester group include a p_nitrophenyl group, an N-hydroxysuccinimide group, a succinic acid imide group, a phthalic acid imide group, and a 5_norbornene-1,3-dicarboxyimide group. For example, a p-nitrophenyl group is preferably used.

 [0032] In the case of a carrier on which an oligo d T chain is immobilized on the surface of an insoluble carrier, as a more specific combination of the first unit and the second unit, for example, the first unit containing a phosphorylcholine group is 2_ A structure having a methacryloyloxychetyl phosphorylcholine group and an active ester group being a p_nitrophenyl group can be employed.

 When the form of the insoluble carrier is a porous body, the oligo d T chain is immobilized on the pore surface, but even in this case, as a combination of a more specific configuration of the first unit and the second unit The first unit containing a phosphorylcholine group has a 2_methacryloxylchetylphosphorylcholine group, and the active ester group is a p_nitrophenyl group.

[0033] In addition, the polymer substance used in the coating layer on the surface of the insoluble carrier of the present embodiment is a group other than a group derived from a phosphate ester or a carboxylic acid derivative group constituting the hydrophilic part of the phospholipid. Groups may be included. The polymer substance can be a copolymer. Specifically, the polymer substance is preferably a copolymer containing a butyl methacrylate group. By so doing, the polymer substance can be appropriately hydrophobized, and the adsorptivity of the polymer substance to the carrier surface can be more suitably ensured.

[0034] Specifically, the polymer substance is composed of a first monomer having a 2_methacryloyloxychetylphosphorylcholine (MPC) group, p_nitrophenyloxypolyethylene glycol methacrylate (NPMA) Second having a group A copolymer of the monomer and a third monomer having a butyl metallate (BMA) group can be obtained. These copolymers, po I y (MPC-c 0-BMA_co_N PMA) (PMBN), are schematically represented by the following general formula (2).

[0035] [Chemical 4]

[0036] However, in the above general formula (2), a, b, and c are each independently a positive integer. In the general formula (2), the first to third monomers may be block copolymerized, or these monomers may be copolymerized randomly.

 [0037] The copolymer represented by the above general formula (2) immobilizes the oligo d T chain with appropriate hydrophobicity of the high molecular weight material, the property of suppressing non-specific adsorption of proteins and DNA chains, and the like. It is an excellent structure in balance with the properties. For this reason, by using such a copolymer, the surface of the insoluble material is more reliably covered with the polymer material, and nonspecific adsorption to the surface of the carrier coated with the polymer material is suppressed. On the other hand, the oligo d T chain can be reliably immobilized by covalent bonding and introduced onto the carrier.

 [0038] The copolymer represented by the general formula (2) can be obtained by a known polymerization method such as radical polymerization by mixing monomers of MPC, BMA, and NPMA. When the copolymer represented by the general formula (2) is prepared by radical polymerization, for example, solution polymerization is performed in an inert gas atmosphere such as Ar under a temperature condition of 30 ° C to 90 ° C. Can do.

[0039] The solvent used in the solution polymerization is appropriately selected. For example, an alcohol such as methanol, ethanol or isopropanol, an ether such as jetyl ether, or an organic solvent such as black mouth form may be used alone or in combination. Mixed use Can do. Specifically, it can be a mixed solvent in which jetyl ether and black mouth form are in a volume ratio of 8 to 2.

 [0040] As the radical polymerization initiator used in the radical polymerization reaction, those usually used can be used. For example, azo-based initiators such as azobisisoptylonitrile (AIBN), azobisvaleronitryl, lauroyl peroxide, benzoyl peroxide, t_butylperoxyneodecanoate, t_butylper Oil-soluble organic peroxides such as oxypivalate are used.

 [0041] More specifically, using a mixed solvent of 8% to 2 by volume ratio of jetyl ether and black mouth form and AIBN, polymerization was performed in Ar at 60 ° C for about 2 to 6 hours. It can be carried out.

 [0042] In the present embodiment, the example in which the high molecular substance has a third unit including a butyl methacrylate group has been described. However, the first unit including a phosphorylcholine group and the second unit including a carboxylic acid derivative group are included. The polymer material may be a first polymer material, and in addition, a second polymer material having a first unit containing a phosphorylcholine group and a third unit containing a butyl methacrylate group may be included.

 [0043] The first unit of the first polymer substance and the first unit of the second polymer substance may have the same structure or different structures. Further, when the first polymer substance includes a third unit containing a butyl methacrylate group, the third unit of the first polymer substance and the third unit of the second polymer substance have the same structure. It may be a different structure.

[0044] Such a second polymer substance is used as a polymer that suppresses non-specific adsorption of a cage DNA fragment. Such polymers, for example phosphate Horirukorin group 3 0 mole _^0/0, the use of the MPC polymer (NOF Corporation) in which heptyl methacrylate group is contained in a proportion of 7 0 mole _^0/0 Is possible.

[0045] In the case where the polymer substance is composed of the first polymer substance and the second polymer substance, the polymer substances may be mixed. Each polymer substance polymer can be dissolved in ethanol solution, for example, A mixed polymer can be easily obtained by mixing each polymer solution.

 [0046] The carrier having the polymer substance as described above on the surface of the insoluble carrier can be obtained, for example, by applying a liquid containing the polymer substance to a carrier molded into a predetermined shape and drying it. Alternatively, the carrier may be immersed in a liquid containing a polymer substance and dried.

 [0047] In the present embodiment, a carrier having the above-described polymer substance on the surface of an insoluble carrier is further added.

Immobilize the DNA chain (oligo d T chain) containing the oligo d T sequence.

 When the number of bases of the oligo d T chain is in the range of 5 to 50, mRNA can be stably captured, and more preferably 15 to 35.

[0048] A liquid in which the oligo d T chain is dissolved or dispersed is immersed in a container, and the carrier is crushed.

It can be dried after standing, and the oligo d T chain can be immobilized on the surface of the carrier.

[0049] The liquid in which the oligo d T chain is dissolved or dispersed can be, for example, neutral to alkaline, for example, having a pH of 7.6 or more.

[0050] Oligo d Oligo d that was not immobilized on the surface of the carrier after immobilization of T chain

In order to remove the T chain, it may be washed with pure water or a buffer solution.

[0051] After washing, the inactive treatment of the active ester on the surface of the carrier other than the oligo d T chain immobilized thereon is performed with an alkyl compound or a compound having a primary amino group.

 [0052] Examples of the alkaline compounds include sodium hydroxide, hydroxy hydroxide, sodium carbonate, sodium bicarbonate, disodium hydrogen phosphate, calcium hydroxide, magnesium hydroxide, sodium borate, hydroxide Lithium, phosphoric acid lithium and the like can be used.

[0053] —Compounds having a secondary amino group include glycine, 9_amino aquaazine, aminobutanol, 4-aminobutyric acid, aminocaprylic acid, aminoethanol, 5-amino2,3-dihydro-1,4_pen Tanol, aminoethanethiol hydrochloride, aminoethanethiol sulfate, 2_ (2-aminoethylamino) ethanol, dihydrogen phosphate 2_aminoethyl, aminoethyl hydrogensulfate, 4 -(2_aminoethyl) morpholine, 5-aminofluorescein, 6_aminohexanoic acid, aminohexylcellulose, p-aminohippuric acid, 2-amino_2-hydroxymethyl_1,3_propane Diol, 5-Aminoisophthalic acid, Aminomethane, Aminophenol, 2-Aminooctane, 2-Aminooctanoic acid, 1-Amino2_propanol, 3-Amino-1-propanol, 3-Aminopropene, 3-Aminopropionitrile, Amino Viridine, 11_aminoundecanoic acid, aminosalicylic acid, aminoquinoline, 4-aminophthalonitrile, ₃ -aminophthalimide, p-aminopropiophenone, aminophenylacetic acid, aminonaphthalene and the like can be used. Of these, aminoethanol and glycine are preferably used.

 [0054] In addition, an amino group is preferably introduced into the oligo d T chain to be immobilized on the carrier in order to enhance the reactivity with the active ester group. Since the amino group is highly reactive with the active ester group, the oligo d T chain can be efficiently and firmly attached to the surface of the carrier by using an oligo d T chain with an amino group introduced at the 5 'end. Can be immobilized.

 [0055] As described above, an mRNA capture carrier having an oligo dT chain immobilized on its surface can be obtained.

 When a porous material is used as the insoluble carrier, a carrier having oligo dT chains immobilized on the pore surface can be obtained.

[0056] A method for purifying mRNA by the carrier of the present embodiment will be described.

 This method uses the carrier for capturing mRNA of this embodiment,

 (a) supplying a sample solution containing mRN A to the m R N A capture carrier,

(b) a step of hybridizing mRNA and oligo d T chain,

 (c) supplying cleaning solution and cleaning,

 It is characterized by including.

 This method may further include the step of (d) eluting the hybridized mRNA.

[0057] In step (a), first, a specimen containing mRNA is prepared. The sample is not particularly limited as long as it contains mRNA with poly A-til. For example, animal-derived cells, plant-derived cells, cells derived from bacteria and fungi can be used. Tissue and blood can also be used. Next, prepare a sample solution from the prepared sample containing the RNA strand. The sample solution can be prepared by dissolving or suspending the sample in a solvent. Preferably, a guanidine isothiocyanate system or a SDS_protease system cell lysate is used. Thus, cells and tissues can be solubilized and prepared. Subsequently, the sample solution is supplied to the above-mentioned mRNA capture carrier. For example, if the carrier is in the form of a container, dispense it into the container. In the case where the form of the carrier is a porous body, the carrier is allowed to permeate into the porous body, or left in the porous body.

 [0058] Next, in step (b), the mRNA and oligo dT chain are hybridized.

 On the polymer substance according to the present embodiment, mRNA is efficiently hybridized, and mRNA can be efficiently captured on the carrier of the present embodiment. More specifically, the oligo d T chain introduced on the surface of the insoluble carrier and the poly A-til part of mRNA are hybridized. On the polymer material used in this embodiment, hybridization between the polyA-til part of mRNA and the oligo dT chain occurs with high efficiency, and a high capture amount of mRNA can be secured. it can.

 Step (b) may comprise incubation at a predetermined temperature, for example 37 ° C.

 [0059] Subsequently, in the step (c), a cleaning liquid is supplied to the carrier to perform cleaning.

 As the cleaning solution, for example, autoclaved ultrapure water can be used.

After capturing the mRNA in step (b), the carrier can be washed by immersing the carrier in a washing solution and moving back and forth inside the porous body. As a result, RNA strands other than the mRNA strand hybridized from the surface of the carrier of the present embodiment, DN A chain, protein, lipid etc. can be removed.

 [0060] When the step (d) is performed, the heated washing water is used in the step (c). The temperature of the wash water can be, for example, 90 ° C.

 By so doing, the RNA and DNA strand after hybridization can be easily dissociated on the polymer used in the present embodiment, and mRNA can be eluted by washing and recovered.

 [0061] As described above, by using the mRNA capturing carrier of the present invention, mRNA in a biological sample can be efficiently captured by a simple operation. Alternatively, mRNA can be dissociated from oligo dT and eluted.

[0062] In the following manner, mRNA was captured using an mRNA capturing carrier comprising an insoluble carrier having a polymer substance corresponding to the present embodiment on its surface. As the form of the insoluble carrier, a porous body and a container (here, a sampling tube) were used. The results were compared using a conventional insoluble carrier with an aldehyde-treated surface.

 (Preparation of PMB N treatment carrier)

Fine particles of porous polyethylene resin were supplied into the mold and subjected to pressure thermoforming to obtain a spherical porous body having a diameter of 3 mm. This porous material is mixed with 2_methacryloyloxysityl phosphorylcholine monobutyl methacrylate _ p-nitrophenyloxycarboxyl polyethylene glycol methacrylate copolymer (po I y (MP C-co-BMA-coN PMA) (PMB N ) Each group was immersed in 0.5% ⁰ / o ethanol solution of 25: 74: 1) in mol%. After leaving for 30 minutes, the excess solution is removed by centrifugation, drying is performed, and the surface of the pores is made of plastic with a phosphorylcholine group and a polymer substance having a p-nitrophenyl group as the active ester group. A porous material (PMBN-treated carrier) was obtained.

 [0063] (Preparation of aldehyde-treated carrier)

Supply fine polyethylene resin particles into the mold, pressurize and heat-mold, diameter 3 A spherical porous body of mm was obtained. The molded product was subjected to hydrophilic treatment on the surface of the pores by low-temperature oxygen plasma treatment. Next, the r- amino propyltrimethoxysilane E butoxy silane as aminoalkyl silanes prepared which is dissolved at a concentration of 5 weight _^0/0 in methanol as Amino group introduction treatment solution, 2 hours in this solution Hita潰Thereafter, this solution was removed by centrifugation, immersed in ultrapure water and allowed to stand, and then the substrate was removed by centrifugation and dried. Glutaraldehyde is dissolved in PBS (-) (calcium-free phosphate buffered saline, Phospate Buffered Sline) at a concentration of 2% by weight to prepare a glutaraldehyde solution, which is treated with aminoalkylsilane. The carrier was immersed in a glutaraldehyde solution and allowed to stand for 4 hours, after which the solution was removed by centrifugation, immersed in ultrapure water, washed and dried. As a result, a porous body (aldehyde-treated carrier) having an aldehyde group on the pore surface was obtained.

 [0064] (Preparation of PMB N-treated tube)

The inner surface of a polypropylene sampling tube (Fukae Chemical Co., Ltd., capacity 1.5 ml) is 2 _methacryloyloxysethyl phosphorylcholine monobutyl acrylate _p—ditrophenyloxypolyethylene glycol methacrylate copolymer (po I y MPC—co—BMA—co—NPMA) (PMBN), each group is dispensed with 0.5 weight ⁰ / o ethanol solution of 25: 74: 1) at ⁰ / o mole and left for 30 minutes After that, the sample is sucked to remove excess solution, dried, and a sampling tube (PMBN treatment) into which a polymer substance having a phosphorylcholine group and p_nitrophenyl group as an active ester group is introduced on the inner surface of the tube Tube).

 [0065] (Production of aldehyde group-treated tube)

A polypropylene sampling tube (Fukae Kasei volume 1.5 ml) was hydrophilized by low-temperature oxygen plasma treatment. Next, a solution prepared by dissolving monoaminopropyl triethoxysilane as an aminoalkyl silane in methanol at a concentration of 5% by volume is prepared as an amino group introduction treatment solution, and this solution is dispensed into a tube for 2 hours. After standing, remove the solution by suction, and add ultrapure water. After dispensing and leaving, ultrapure water was removed by suction and dried. Glutaraldehyde is dissolved in PBS (1) at a concentration of 2% by volume to prepare a glutaraldehyde solution, dispensed into a tube treated with an aminoalkylsilane, and left for 4 hours, then the solution by suction The ultrapure water was dispensed into the tube, washed, sucked and dried. As a result, a sampling tube (aldehyde-treated tube) having an aldehyde group on the inner surface of the container was obtained.

[0066] (Immobilization of oligo d T chain to porous body)

 5'-end modified with amino group, TTTTTTTTTTTTTTTTTTTTT TTTTTTTTT (30 bases) oligo d T chain is dissolved using 0.25M carbonate buffer (pH 9.0), and 10 ~ oligo T solution Was prepared. Each of the porous bodies was immersed in this solution, allowed to stand for 10 minutes, and then dried to immobilize oligo dT chains. Thereafter, each carrier was subjected to a blocking treatment.

 [0067] (Immobilization of oligo d T chain to sampling tube)

 Dissolve the oligo d T chain (d T primer) of the sequence TTTTTTTTTTTTTTTTTTTTTTTTTTTTT (30 bases) modified with an amino group at the 5 'end using 0.25 M carbonate buffer (pH 9.0). M oligo T solution was prepared. This solution was dispensed into a sampling tube, allowed to stand for 10 minutes, and then dried to immobilize the oligo dT chain. Thereafter, each tube was subjected to a blocking treatment.

 [0068] (Preparation of RNA solution from cells)

In a petri dish for cell culture with a diameter of 35 mm, cultivate He La cells until confluent. After removing the medium, wash gently with PBS (-), discard the RNA extraction medium from adherent cells on ice, and wash several times with PBS. A 3 ml I GTC solution was placed in the dish, and the cells were lysed by scraping the cells with a cell scraper and collected in a sampling tube with a pipette. The collected solution was put into and out of a syringe barrel equipped with an 18 G needle, mixed well, and subjected to an evaluation experiment as an RNA solution. [0069] (Evaluation of mRNA capture in porous media)

 Each porous body to which the oligo d T chain was immobilized was cut at the tip of a dispenser chip having a capacity of 1 ml, and each porous body was fitted and fixed.

 Dispense approximately 100 〃 of the above RNA solution into a 1.5 ml sampling tube, hold the porous material fixed to the tip of the dispenser tip in the RNA solution, and perform RN The solution A was repeatedly taken in and out of the porous body, and then left at room temperature with the RNA solution retained in the porous body. After that, put PBS (-) in a separate sampling tube for about 500〃, hold the above porous body in it, and take out PBS (-) in and out of the porous body by dispenser operation. Was washed.

 In a separate sampling tube, take 10 On I of autoclaved ultrapure water, heat to 90 ° C, hold the washed porous body, and perform lyoclave treatment with a dispenser operation. The ultrapure water was repeatedly taken in and out, and the mRNA retained in the porous body was washed out and collected.

 The absorbance at 260 nm of the collected solutions was compared, and the amount of mRNA in the collected solutions was compared for each porous material. The results are shown in Table 1.

[0070] (Evaluation of mRNA capture in sampling tube)

 The above RNA solution was dispensed into each tube to which oligo d T chain had been immobilized in an amount of 1 O O I and allowed to stand at room temperature for 15 minutes while retaining the RNA solution. Thereafter, the RNA solution was removed by suction, and then washed twice with PBS (-). 1 O O I of autoclaved ultrapure water was taken, heated to 90 ° C, and a solution containing mRNA was collected in another sampling tube.

 The absorbance of the collected solutions at 260 nm was compared, and the amount of mRNA in the collected solutions was compared for each tube. The results are shown in Table 2.

[0071] 1

[0072] Ho 2

[0073] In the PMBN-treated carrier and the PMBN-treated tube surface-treated with PMBN, which is a type of polymer material according to the present embodiment, the amount of the collected mRNA was much higher, whereas in the aldehyde-treated carrier, The amount of mRNA collected was very small. Therefore, with an insoluble carrier having PMBN, mRNA can be purified and fractionated with very high efficiency compared to the conventional carrier surface.

 [0074] Embodiments of the present invention are listed below.

 (1) Carboxyl formed by bonding a first unit having a group derived from a phosphate ester constituting the hydrophilic part of a phospholipid on the pore surface of a porous body and an electron-attracting substituent bonded to a carbonyl group A polymer substance containing a second unit having an acid-derived group, and a DNA strand containing an oligo dT sequence bound to all or a part of the carboxylic acid-derived group is bound at the 5 ′ end of the oligo dT sequence. A carrier for capturing mRNA.

 (2) The mRNA capture carrier according to (1), wherein the oligo dT sequence has 5 to 50 bases.

(3) m described in (1) or (2), wherein the material of the porous body is plastic. RNA capture carrier.

 (4) Using the mRNA capture carrier according to any one of (1) to (3),

 (a) supplying a sample solution containing an mRNA chain to the mRNA extraction carrier;

 (b) a step of hybridizing mRNA and oligo d T chain,

 (c) supplying cleaning solution and cleaning,

A method for purifying mRNA comprising the steps of:

 (5) The method for purifying mRNA according to (4), further comprising (d) a step of eluting the hybridized mRNA.

Claims

The scope of the claims  [1] A first unit having a group derived from a phosphate ester constituting the hydrophilic part of a phospholipid and a carboxylic acid derivative group formed by bonding an electron-withdrawing substituent to a carbonyl group on the surface of the insoluble carrier. And a DNA containing an oligo dT sequence in all or part of the force-derived rubonic acid-derived group is bound to the 5 ′ end of the oligo dT sequence. A carrier for capturing mRNA.  [2] The support for capturing mRNA according to claim 1, wherein the oligo dT sequence has 5 to 50 bases.  [3] The carrier for capturing mRNA according to claim 1 or 2, wherein the insoluble carrier is a container or a plate having a plurality of wells. [4] The support for capturing mRNA according to claim 1 or 2, wherein the form of the insoluble support is a porous material.  5. The mRNA capture carrier according to any one of claims 1 to 4, wherein the material of the insoluble carrier is plastic. [6] Using the carrier for capturing mRNA according to any one of claims 1 to 5,  (a) supplying a sample solution containing mRNA to the mRNA capture carrier; (b) Hybridizing mRNA and oligo d T sequence DNA (c) supplying cleaning solution and cleaning,  A method for purifying mRNA comprising the steps of: [7] The method further comprising the step of (d) eluting the hybridized mRNA. 6. The method for purifying mRNA described in 6. 8. The method for purifying mRNA according to claim 7, wherein the sample solution containing mRNA is a solution in which cells are solubilized.