WO2011162326A1 - Solution de conservation de brin d'arn - Google Patents

Solution de conservation de brin d'arn Download PDF

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WO2011162326A1
WO2011162326A1 PCT/JP2011/064366 JP2011064366W WO2011162326A1 WO 2011162326 A1 WO2011162326 A1 WO 2011162326A1 JP 2011064366 W JP2011064366 W JP 2011064366W WO 2011162326 A1 WO2011162326 A1 WO 2011162326A1
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rna
strand
rna strand
solution
pcr
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PCT/JP2011/064366
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Japanese (ja)
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寛一 中川原
徹 八重樫
啓史 齋藤
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株式会社日本遺伝子研究所
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Publication of WO2011162326A1 publication Critical patent/WO2011162326A1/fr

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    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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

Definitions

  • the present invention relates to a storage solution for RNA strands, a method for producing a storage solution for RNA strands, a method for stabilizing RNA strands, and the like.
  • the measurement of the expression level of RNA is important in diagnosing a predetermined state such as a disease and conducting an experiment.
  • One method for measuring the expression level of RNA is RT-PCR.
  • RT-PCR is performed by (i) reverse transcription (RT) reaction for synthesizing cDNA from RNA, and (ii) amplification reaction of synthesized cDNA.
  • RT reverse transcription
  • an RNA preparation is required as a control.
  • a reliable RNA preparation that can be used for RT-PCR is provided to clinical sites. I could't. This is because RNA degrades even in a frozen state at ⁇ 20 ° C., and long-term storage is difficult. Therefore, when measuring the expression level of the target RNA strand, the conventional RT-PCR performs the RT reaction and amplification reaction (PCR) separately, and after the RT reaction ends, only the amplification reaction is verified. Used the product.
  • RNA sample it is possible to verify a one-step RT-PCR in which an RT reaction and an amplification reaction are performed simultaneously.
  • Patent Document 1 and Non-Patent Document 1 describe RNase inhibitors such as aurintricarboxylic acid (hereinafter sometimes referred to as ATA as needed).
  • Non-Patent Documents 1 and 2 describe aurintricarboxylic acid (ATA) as an RNase inhibitor.
  • Non-Patent Document 3 describes that sodium citrate can be used as a chelating agent that suppresses hydrolysis of RNA bases.
  • RNA preparation As described above. The reason is that although substances that suppress RNA degradation are known as described above, these substances are not always capable of sufficiently inhibiting the degradation of RNA preparations. Substances that suppress RNA degradation are often strong protein denaturing agents, and when RNA solutions containing protein denaturing agents are used for RT reactions and amplification reactions that are enzymatic reactions, the protein denaturing agents in the solution Since these reactions are inhibited, there is a problem that RNA in such a solution cannot be used as an RNA preparation for verifying a reaction such as RT-PCR. Therefore, the development of a method for stabilizing an RNA strand that realizes the supply of an RNA preparation for verifying a reaction such as RT-PCR is eagerly desired.
  • the present invention has been made in view of the above, and an object thereof is to realize supply of an RNA sample for verifying a reaction such as RT-PCR.
  • the present inventors have succeeded in finding a component that can stabilize an RNA strand and cannot inhibit a reaction such as RT-PCR, and has completed the present invention.
  • RNA strand preservation solution containing an RNA strand to be preserved and a single-stranded RNA capable of stabilizing the RNA strand.
  • the storage solution according to [1], wherein the single-stranded RNA capable of stabilizing the RNA strand is phage RNA.
  • the preservation solution according to [1], wherein the RNA strand to be preserved is an RNA preparation.
  • the preservation solution according to [1], wherein the RNA strand to be preserved is a single-stranded RNA.
  • the preservation solution according to [1], wherein the RNA strand to be preserved is total RNA.
  • RNA strand storage solution Storing the RNA strand storage solution in a liquid state; The method, wherein the RNA strand storage solution is an RNA strand storage solution containing an RNA strand to be stored and a single-stranded RNA capable of stabilizing the RNA strand.
  • a method for producing an RNA strand storage solution comprising obtaining a strand storage solution.
  • Kit including the following (a) and (b): (A) an RNA strand storage solution containing an RNA strand to be stored and a single-stranded RNA capable of stabilizing the RNA strand; and (b) a component for the synthesis reaction of the target DNA strand.
  • the component for synthesis reaction of the target DNA strand is a component for reverse transcription reaction or a component for reverse transcription reaction-amplification reaction of target DNA strand.
  • the kit according to [10], wherein the constituent element for the synthesis reaction of the target DNA strand is a reagent for RT-PCR.
  • a method for producing a target DNA strand Subjecting the RNA strand to a synthesis reaction of the target DNA strand, A method wherein the RNA strand is an RNA strand to be preserved in a preservation solution of the RNA strand, comprising an RNA strand to be preserved and a single-stranded RNA capable of stabilizing the RNA strand.
  • RNA strand preservation solution comprising an RNA strand to be preserved and 0.1 to 2.5 ⁇ M aurintricarboxylate.
  • save solution of this invention can stabilize RNA strand, it is useful for preservation
  • the preservation solution of the present invention is also useful as an RNA preparation in the synthesis reaction of the target DNA strand because it cannot substantially inhibit the synthesis reaction of the target DNA strand (eg, RT reaction, PCR, RT-PCR). .
  • the preservation solution of the present invention is excellent in that a stabilized RNA preparation can be supplied in the form of an aqueous solution rather than freeze-drying.
  • RNA preparations are used for testing purposes such as clinical tests, if the RNA preparation can be supplied in an aqueous solution rather than lyophilized, the step of dissolving the RNA preparation in an aqueous solution can be omitted.
  • the inspection can be easily performed.
  • the RNA sample concentration based on inadvertent human error due to carelessness eg, mix-up of a solution for dissolving the RNA sample and variation in the amount of solution added
  • Error Omitting such a process is particularly advantageous from the point of view of quality control when the product is supplied globally and used by those unfamiliar with molecular biology techniques (eg, product use in developing countries). is there.
  • the preservation solution of the present invention can be used to supply RNA preparations stabilized in an aqueous solution to the world. This is because the stock solution of the present invention can stabilize the RNA preparation over a long period of time, so that degradation of the RNA preparation can be suppressed even when the transport period is long.
  • FIG. 1 is a diagram showing a PCR amplification curve for single-stranded RNA (human TERT gene) stored for 7 weeks at 4 ° C. in the presence of each component.
  • FIG. 2 is a diagram showing a PCR amplification curve for single-stranded RNA (human TERT gene) stored for 7 weeks at room temperature in the presence of each component.
  • FIG. 3 is a diagram showing Cp value change with time (real-time PCR by an intercalator method) of single-stranded RNA (human TERT gene) stored at 4 ° C. in an aqueous MS2 RNA solution.
  • FIG. 1 is a diagram showing a PCR amplification curve for single-stranded RNA (human TERT gene) stored for 7 weeks at 4 ° C. in the presence of each component.
  • FIG. 2 is a diagram showing a PCR amplification curve for single-stranded RNA (human TERT gene) stored for 7 weeks at room temperature in the presence of each component.
  • FIG. 4 is a diagram showing Cp value change over time (real-time PCR by an intercalator method) of single-stranded RNA (human TERT gene) stored at 4 ° C. in a mixed aqueous solution of MS2 RNA and ammonium aurintricarboxylate.
  • FIG. 5 is a graph showing Cp value change with time (real-time PCR by an intercalator method) of single-stranded RNA (human TERT gene) stored at room temperature in an MS2 RNA aqueous solution.
  • FIG. 5 is a graph showing Cp value change with time (real-time PCR by an intercalator method) of single-stranded RNA (human TERT gene) stored at room temperature in an MS2 RNA aqueous solution.
  • FIG. 6 is a diagram showing Cp value change over time (real-time PCR by an intercalator method) of single-stranded RNA (human TERT gene) stored at room temperature in a mixed aqueous solution of MS2 RNA and ammonium aurintricarboxylate.
  • FIG. 7 is a diagram showing the Cp value change with time (real-time PCR using a hydrolysis probe) of single-stranded RNA (human TERT gene) stored at 4 ° C. in an aqueous MS2 RNA solution.
  • FIG. 8 is a diagram showing Cp value change over time (real-time PCR using a hydrolysis probe) of single-stranded RNA (human TERT gene) stored at 4 ° C.
  • FIG. 9 is a diagram showing Cp value change with time (real-time PCR using a hydrolysis probe) of single-stranded RNA (human TERT gene) stored at room temperature in an MS2 RNA aqueous solution.
  • FIG. 10 is a diagram showing Cp value change with time (real-time PCR using a hydrolysis probe) of single-stranded RNA (human TERT gene) stored at room temperature in a mixed aqueous solution of MS2 RNA and ammonium aurintricarboxylate.
  • FIG. 11 is a diagram showing the change over time of the Cp value of total RNA stored at 4 ° C.
  • FIG. 12 is a graph showing the Cp value change with time of total RNA stored at room temperature in the presence of each component.
  • FIG. 13 is a diagram showing a PCR amplification curve for single-stranded RNA (human TERT gene) stored in an ATA (ammonium salt) aqueous solution at 4 ° C. or room temperature for 2 weeks.
  • ATA ammonium salt
  • the present invention provides an RNA strand storage solution (or RNA strand solution; the same shall apply hereinafter).
  • the preservation solution of the present invention contains an RNA strand to be preserved and components capable of stabilizing the RNA strand.
  • the RNA strand to be stored is a storage target to be stabilized by a component capable of stabilizing the RNA strand. Examples of components that can stabilize the RNA strand include single-stranded RNA that can stabilize the RNA strand, and aurintricarboxylate.
  • the RNA strand to be stored is composed of ribonucleotides that are components of the RNA strand.
  • ribonucleotides that are components of RNA strands to be stored include, for example, adenosine 5′-phosphate having adenine (A) as a nucleobase as a main ribonucleotide and guanosine 5 ′ having guanine (G) as a nucleobase.
  • uridine 5'-phosphate having uracil (U) as a nucleobase.
  • ribonucleotides include, for example, inosine, pseudouracil, 5-methylcytosine, 2-aminopurine, 2,6-diaminopurine, pyrrolocytosine, and 5-methyluracil.
  • the RNA strand to be stored may be single-stranded or double-stranded.
  • the RNA strands to be preserved are RNA strands composed of the same type of ribonucleotides (ribonucleotide homopolymers), but are RNA strands composed of different types of ribonucleotides (ribonucleotide heteropolymers). There may be.
  • modified ribonucleotide refers to an unmodified ribonucleotide or a modification of at least one of a nucleobase moiety, a sugar moiety and a phosphate binding moiety.
  • modified ribonucleotides include ribonucleotides containing a nucleobase moiety substituted with a substituent, ribonucleotides containing a sugar moiety substituted with a substituent, and phosphate linkages exchanged with other linkages. And ribonucleotides containing combinations of these modifications.
  • substituents examples include an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, an amino group, a halogen atom (eg, a fluorine atom, a bromine atom, a chlorine atom, an iodine atom), Examples thereof include a hydroxyl group, a carboxyl group, an oxo group ( ⁇ O), a thio group ( ⁇ S), and a nitro group.
  • the number of substituents that can be bonded to the nucleobase moiety or sugar moiety can be 1 to 3, preferably 1 or 2, and more preferably 1. When the phosphate binding moiety is exchanged for other bonds, such other bonds include phosphorothioate bonds, amide bonds, methylphosphonate bonds, methylphosphotriester bonds.
  • alkyl group examples include linear or branched alkyl having 1 to 12 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl.
  • C 1 -C 10 alkyl such as heptyl, octyl, nonyl, decyl and the like, with C 1 -C 6 alkyl being preferred.
  • alkenyl group a linear or branched alkenyl having 2 to 12 carbon atoms, for example, allyl, crotyl, 2-pentenyl, 3-hexenyl C 2 ⁇ C 10 is alkenyl and the like, such as, C 2 ⁇ C 6 alkenyl is preferred.
  • alkynyl group linear or branched alkynyl having 2 to 12 carbon atoms, such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-pentynyl, C 2, such as 3-hexynyl ⁇ C
  • C 2 -C 6 alkynyl is preferred.
  • cycloalkyl group examples include cyclic cycloalkyl having 3 to 12 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
  • aryl group examples include aryl having 6 to 12 carbon atoms such as phenyl, 1-naphthyl, and 2-naphthyl.
  • Aralkyl groups include aralkyl having 7 to 12 carbon atoms, such as phenyl-C 1 -C 4 alkyl (eg, benzyl, phenethyl).
  • acyl group examples include acyl having 2 to 12 carbon atoms, such as alkanoyl having 2 to 4 carbon atoms (eg, acetyl, propionyl, butyryl, isobutyryl), or aroyl having 6 to 10 carbon atoms (eg, benzoyl, toluoyl, etc.) Is mentioned.
  • alkanoyl having 2 to 4 carbon atoms eg, acetyl, propionyl, butyryl, isobutyryl
  • aroyl having 6 to 10 carbon atoms eg, benzoyl, toluoyl, etc.
  • amino group an amino which may be mono- or di-substituted with a substituent selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl and acyl, for example, amino (—NH 2 ), Examples include methylamino and dimethylamino.
  • the RNA strand to be stored may be a naturally derived RNA strand.
  • RNA strands derived from nature include natural RNA strands that can be prepared from natural sources (eg, animals such as mammals, plants, insects, microorganisms, viruses).
  • the RNA chain derived from nature may be total RNA, messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), nuclear low molecular RNA (snRNA), or microRNA (miRNA). Good.
  • the RNA strand to be stored may be an artificially synthesized RNA strand.
  • Examples of the artificially synthesized RNA chain include siRNA, probes, and RNA vectors.
  • the number of ribonucleotides in the RNA strand to be stored is not particularly limited as long as it is 2 or more. For example, 5 or more, 10 or more, 16 or more, 20 or more, 30 or more, 50 or more, It may be 100 or more, 200 or more, or 500 or more.
  • the number of ribonucleotides in the RNA strand is not particularly limited, and may be, for example, less than 100,000, less than 50,000, less than 10,000, or less than 5,000.
  • RNA strand to be stored contained in the storage solution of the present invention is a single (ie, one type) RNA strand, a plurality (eg, 2, 3, 5, 10, 50). RNA strands of 100 species, 100 species, 200 species, 500 species, or 1000 species or more).
  • the RNA strand to be stored is used as an RNA preparation, for example.
  • examples of the RNA strand to be stored include an RNA strand of a housekeeping gene, or other RNA strands that can be used as a standard in an assay using an RNA strand.
  • examples of the RNA strand of the housekeeping gene include RNA strands encoding GAPDH, ⁇ -actin, ⁇ 2-microglobulin, HPRT1, ⁇ -tubulin, L19, cyclophilin A, and transferrin R.
  • RNA strands that can be used as preparations in assays utilizing RNA strands include, for example, TERT, CK-19, HCV, HIV, BCR / ABL (eg, Major BCR / ABL), WT-1, RNA
  • An RNA strand encoding a virus eg, influenza virus, dengue virus, West Nile virus
  • the RNA strand to be stored is preferably a human-derived RNA strand from the viewpoint of clinical examination.
  • RNA preparation for reverse transcription (RT) reaction or amplification reaction (eg, RT-PCR) of a target DNA strand involving RT reaction.
  • RT-PCR is performed by (i) reverse transcription (RT) reaction for synthesizing cDNA from RNA, and (ii) amplification reaction of synthesized cDNA.
  • RT reverse transcription
  • amplification reaction of synthesized cDNA.
  • RT-PCR it is possible to provide an RNA preparation that can verify the RT reaction and both the RT reaction and the amplification reaction.
  • provision of RNA preparations enables verification of one-step RT-PCR in which RT reaction and amplification reaction are performed simultaneously.
  • the RNA strand to be stored may be total RNA when used as an RNA preparation in an assay for measuring an RNA expression profile (eg, microarray, Northern blotting).
  • the RNA strand (total RNA) to be stored is used for the quality control of the assay in the same manner as the control serum generally used for the quality control of the clinical test.
  • the component capable of stabilizing the RNA strand is a single-stranded RNA capable of stabilizing the RNA strand.
  • the single-stranded RNA that can stabilize the RNA strand is a single-stranded RNA that can suppress degradation of the RNA strand, and is an RNA strand that is different from the RNA strand to be stored as a substance. Accordingly, the preservation solution of the present invention may contain at least two RNA strands.
  • the single-stranded RNA that can stabilize the RNA strand may be purified.
  • Commercially available single-stranded RNA for RNA assay may be used as the single-stranded RNA that can stabilize the RNA strand.
  • Such a single-stranded RNA can be suitably used because it is RNase-free.
  • Single-stranded RNA that can stabilize RNA strands is RNA strands composed of different types of ribonucleotides (ribonucleotides), even if they are RNA strands composed of the same type of ribonucleotides (ribonucleotide homopolymer). Heteropolymer).
  • Single-stranded RNA that can stabilize the RNA strand is composed of ribonucleotides as described above.
  • the single-stranded RNA that can stabilize the RNA strand may be a naturally derived RNA strand.
  • Examples of such single-stranded RNA include natural single-stranded RNA that can be prepared from natural sources (eg, animals such as mammals, plants, insects, microorganisms, phages and other viruses).
  • the single-stranded RNA may be an artificially synthesized RNA strand.
  • the single-stranded RNA may be derived from a different source from the RNA strand to be stored. Therefore, the preservation solution of the present invention comprises an RNA strand to be preserved derived from a first source (eg, an animal species such as a human) and a second source (eg, phage) different from the first source.
  • the preservation solution of the present invention may be one in which one of the RNA strand to be preserved and the single-stranded RNA capable of stabilizing the RNA strand is artificially synthesized and the other is naturally derived.
  • a single-stranded RNA capable of stabilizing an RNA strand is a single-stranded RNA having a self-replicating ability that can be amplified by infecting a predetermined organism (eg, microorganism) from the viewpoint of ease of procurement (eg, , Single-stranded RNA having a replication origin).
  • single-stranded RNA examples include viral RNA such as phage RNA (eg, MS2 RNA) and vector RNA.
  • viral RNA such as phage RNA (eg, MS2 RNA)
  • vector RNA Preferable examples of the single-stranded RNA that can stabilize the RNA strand include phage RNA (eg, MS2 RNA).
  • the number of ribonucleotides in the single-stranded RNA that can stabilize the RNA strand is not particularly limited as long as it is a number that can stabilize the RNA strand. For example, 16 or more, 30 or more, 50 or more, 100 One or more, 200 or more, 500 or more, 1000 or more, or 2000 or more may be sufficient.
  • the number of ribonucleotides in the single-stranded RNA that can stabilize the RNA strand is also not particularly limited, but for example, less than 200,000, less than 100,000, less than 20,000, or less than 10,000 There may be.
  • save solution of this invention is single (namely, one kind) single stranded RNA, it is plural (for example, 2 types, 3 types). Although it may be a single-stranded RNA, it is preferably a single-stranded RNA from the viewpoint of simplification of contained components and simplification of preparation of the preservation solution of the present invention.
  • the preservation solution of the present invention may contain a single-stranded RNA capable of stabilizing the RNA strand at a concentration sufficient to suppress the degradation of the RNA strand to be preserved.
  • a concentration may vary depending on the type and size of single-stranded RNA used (number of ribonucleotides) and the presence or absence of another stabilizer described later. It can be determined as appropriate.
  • the concentration of single-stranded RNA that can stabilize the RNA strand is not particularly limited as long as the RNA strand can be stabilized, but for example 0.1 to 1000 ng / ⁇ L, preferably 0.5 to 500 ng / ⁇ L, more preferably 1.0 to 100 ng / ⁇ L, most preferably 5.0 to 50 ng / ⁇ L.
  • the total concentration of the plurality of single-stranded RNAs may be within the above-mentioned concentration range.
  • the component capable of stabilizing the RNA strand is aurintricarboxylate.
  • Aurin tricarboxylate is used as an RNase inhibitor at high concentrations (see Patent Literature 1 and Non-Patent Literatures 1 and 2), but can be used as an RNase inhibitor at low concentrations (eg, concentrations below 10 ⁇ M), and The use of solutions containing aurintricarboxylate and RNA strands in reverse transcription reactions has not been reported.
  • Examples of the salt of aurintricarboxylic acid include metal salts, inorganic salts, and organic salts.
  • Examples of the metal salt include monovalent metal salts such as sodium salt and potassium salt, and divalent metal salts such as calcium salt and magnesium salt.
  • Examples of inorganic salts include ammonium salts.
  • Examples of the organic salt include an ammonium salt substituted with an alkyl group. Examples of ammonium salts substituted with alkyl groups include monoalkyl ammonium salts, dialkyl ammonium salts, trialkyl ammonium salts, and tetraalkyl ammonium salts.
  • the alkyl group in the ammonium salt substituted with an alkyl group is not particularly limited in carbon number, but may be an alkyl group having 1 to 6 carbon atoms.
  • the alkyl group having 1 to 6 carbon atoms is linear or branched and includes, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, sec-pentyl, Examples include tert-pentyl, hexyl, and isohexyl.
  • the preservation solution of the present invention can suppress degradation of the RNA strand to be preserved contained in the preservation solution of the present invention by using the aurin tricarboxylate, and the preservation solution of the present invention can perform reverse transcription reaction and amplification of the target DNA strand.
  • it may be contained at a concentration at which these reactions cannot be inhibited.
  • concentration depends on the type and size of the single-stranded RNA used (number of ribonucleotides), the presence or absence of another stabilizer described later, and the preservation of the present invention added to the reverse transcription reaction solution or amplification reaction solution. Although it may vary depending on the amount of the solution, for example, 0.1 to 2.5 ⁇ M, preferably 0.2 to 2.0 ⁇ M, more preferably 0.5 to 1.5 ⁇ M, and most preferably 0.8 to 1. 2 ⁇ M.
  • the preservation solution of the present invention may further contain another stabilizer in addition to the single-stranded RNA or / and aurintricarboxylate capable of stabilizing the RNA strand, which is a component capable of stabilizing the RNA strand.
  • Another stabilizer includes, for example, citrate.
  • the other stabilizer is a salt
  • examples of the salt include the salts described above, for example, metal salts such as sodium, and inorganic salts such as ammonium.
  • the solvent in which the RNA strand and the component capable of stabilizing the RNA strand are dissolved is an aqueous solution.
  • the aqueous solution include water (eg, sterile distilled water) and a buffer solution.
  • the buffer solution include TE (Tris-EDTA) buffer solution, hydrochloric acid-potassium chloride buffer solution, glycine-hydrochloric acid buffer solution, citrate buffer solution, acetate buffer solution, citrate-phosphate buffer solution, and phosphate buffer solution.
  • the aqueous solution may or may not be treated with DEPC.
  • the present invention also provides a storage method for the storage solution of the present invention.
  • the storage method of the present invention includes storing the storage solution of the present invention in a liquid state. Storage in a liquid state may be achieved by storing at room temperature.
  • room temperature refers to a temperature of 2 to 40 ° C.
  • JIS Japanese Industrial Standard
  • normal temperature is defined as a temperature of 20 ° C. ⁇ 15 ° C. (5 to 35 ° C.).
  • JIS Japanese Industrial Standard
  • normal temperature is defined as a temperature of 15 to 25 ° C.
  • the storage solution of the present invention may be stored at a temperature of 2 to 40 ° C., but may be stored at a temperature of 5 to 35 ° C. or 15 to 25 ° C.
  • the storage period is not particularly limited as long as a part of the RNA strand to be stored can remain in an undegraded state in the storage solution of the present invention.
  • the degree of the remaining RNA strand to be stored is not particularly limited because it varies depending on the storage period.
  • the RNA strand to be stored is the initial amount of the RNA strand to be stored in the storage solution of the present invention.
  • it can remain undecomposed in an amount of 30% or more, preferably 50% or more, 70% or more, 80% or more, or 90% or more.
  • the degree of the remaining RNA strand to be stored in the storage solution of the present invention can be determined by a method well known in the art (eg, determination of the main peak remaining ratio by TOF / MS detection).
  • the remaining RNA strand to be stored in the storage solution of the present invention can also be confirmed by measuring the Cp value by real-time PCR.
  • the storage period include a period of 1 week or more, 2 weeks or more, 3 weeks or more, 1 month or more, 2 months or more, or 3 months or more. Further, the storage period is not particularly limited, but may be, for example, a period of 1 year or less, 6 months or less, or 4 months or less.
  • the present invention provides a method for producing the preservation solution of the present invention.
  • the production method of the present invention includes a step of allowing an RNA strand to be stored and a component capable of stabilizing the RNA strand to coexist in an aqueous solution.
  • step (1) adding a component capable of stabilizing the RNA strand to the aqueous solution containing the RNA strand to be stored, step (2) in an aqueous solution containing the component capable of stabilizing the RNA strand
  • the RNA strand to be preserved may be added, and the RNA strand to be preserved and the component capable of stabilizing the RNA strand may be simultaneously added to the aqueous solution in step (3).
  • the aqueous solution containing the RNA strand to be stored may be prepared in advance or may be prepared anew. Therefore, when prepared again, in step (1), the RNA strand to be stored is added to an aqueous solution to prepare an aqueous solution containing the RNA strand to be stored, and then the RNA solution is prepared in the prepared aqueous solution. This is done by adding a component capable of stabilizing the chain.
  • an aqueous solution containing a component capable of stabilizing the RNA strand may be prepared in advance, or may be prepared anew. Therefore, when prepared again, the step (2) adds an ingredient capable of stabilizing the RNA strand to the aqueous solution to prepare an aqueous solution containing the ingredient capable of stabilizing the RNA strand, and then the prepared aqueous solution. This is performed by adding an RNA strand to be stored.
  • the simultaneous addition stabilizes the RNA strand to be stored and the RNA strand to be stored by adding a mixture of the RNA strand to be stored and a component capable of stabilizing the RNA strand to an aqueous solution, or This is achieved by adding the resulting components separately into the aqueous solution simultaneously.
  • the production method of the present invention can stabilize the RNA strand to be preserved and the RNA strand. Mixing the ingredients may be included.
  • the aqueous solution used in the production method of the present invention is the same as the aqueous solution described above in the preservation solution of the present invention.
  • the step of allowing the RNA strand to be stored and the component capable of stabilizing the RNA strand to coexist in the aqueous solution can be performed so that the above-described concentration and the like are achieved in the storage solution of the present invention.
  • the production method of the present invention may include adding another component (eg, another stabilizer) to the aqueous solution in addition to the RNA strand to be stored and the component capable of stabilizing the RNA strand.
  • another component eg, another stabilizer
  • the addition of the other components to the aqueous solution can be performed by the same methodology as the above-described steps (1) to (3).
  • the present invention provides a method for stabilizing RNA strands.
  • the stabilization method of the present invention includes stabilizing an RNA strand to be preserved by allowing the RNA strand to be preserved and a component capable of stabilizing the RNA strand to coexist in an aqueous solution.
  • the coexistence of the RNA strand to be stored and the component capable of stabilizing the RNA strand in the aqueous solution can be performed, for example, in the same manner as in the above steps (1) to (3).
  • the aqueous solution used in the stabilization method of the present invention is the same as the aqueous solution described above in the preservation solution of the present invention. Coexistence can be performed such that the concentrations as described above are achieved in the preservation solution of the present invention.
  • the stabilization method of the present invention also includes adding another component (eg, another stabilizer) to the aqueous solution in addition to the RNA strand to be stored and the component capable of stabilizing the RNA strand. Also good.
  • the addition of the other components to the aqueous solution can be performed by the same methodology as the above-described steps (1) to (3).
  • the present invention also provides a kit comprising the stock solution of the present invention.
  • the kit of the present invention includes the following (a) and (b): (A) An RNA strand storage solution containing an RNA strand to be stored and a component capable of stabilizing the RNA strand; and (b) a component for a synthesis reaction of the target DNA strand.
  • the components (a) and (b) included in the kit can be provided in a form isolated from each other, for example, stored in different containers (eg, tubes).
  • the component (a) in the kit of the present invention is the same as the preservation solution of the present invention.
  • the kit of the present invention may further contain a component (c) a DNA or RNA extraction reagent.
  • examples of the component for the synthesis reaction of the target DNA strand include, for example, the component for the reverse transcription reaction of the RNA strand, the reverse transcription reaction—the amplification reaction of the target DNA strand (required in this specification) Depending on the above, constituent elements for “reverse transcription (RT) -amplification reaction” may be mentioned.
  • the RNA strand subjected to the reverse transcription reaction is the above-described RNA strand to be preserved. Therefore, the RNA strand subjected to the reverse transcription reaction may be a naturally derived RNA strand (eg, total RNA, mRNA).
  • kit of the present invention is a kit for reverse transcription reaction of an RNA strand
  • the following (a1) and (b1) are included: (A1) an RNA strand storage solution containing an RNA strand to be stored and a component capable of stabilizing the RNA strand; and (b1) a component for reverse transcription reaction of the RNA strand.
  • the kit of the present invention can be used for the synthesis of the target DNA strand.
  • components for the reverse transcription reaction of RNA strands include reagents and containers used in such reactions.
  • examples of such a component include reverse transcriptase, a primer for reverse transcription reaction, a dNTP mixture, a reaction buffer, and a reaction vessel.
  • the kit of the present invention may be a kit for reverse transcription (RT) -amplification reaction by further including a component for amplification reaction of the target DNA strand.
  • the kit of the present invention includes, for example, the following (a2) and (b2): (A2) an RNA strand storage solution containing an RNA strand to be stored and a component capable of stabilizing the RNA strand; and (b2) a component for reverse transcription reaction of the RNA strand and an amplification reaction of the target DNA strand.
  • the amplification reaction of the target DNA strand is not particularly limited as long as it is an amplification reaction using cDNA synthesized from an RNA strand, and can be used for qualitative or quantitative assays.
  • the amplification reaction of the target DNA strand includes PCR (eg, real-time PCR, multiplex PCR), LAMP (Loop-mediated isometric amplification) (eg, see International Publication No. 00/28082), ICAN (Isothermal) and Chimeric primer-initiated Amplification of Nucleic acids (see, for example, International Publication No. 00/56877) and SMAP (SMart Amplification Process).
  • the kit of the present invention can be used for purposes such as detection, quantification, or cloning.
  • components for the amplification reaction of the target DNA strand include reagents and containers used in such a reaction.
  • such components include a polymerase (eg, a thermostable polymerase), one or more primers (eg, a primer for reverse transcription reaction of an RNA strand, and a primer for amplification reaction of a target DNA strand, Alternatively, a common primer for reverse transcription reaction of RNA strand and amplification reaction of target DNA strand), dNTP mixture, reaction buffer, control template, and reaction vessel.
  • the reverse transcription (RT) reaction and the amplification reaction may be performed separately or simultaneously.
  • the primer for the reverse transcription reaction of the RNA strand and the primer for the amplification reaction of the target DNA strand may be different or may be common.
  • the kit of the present invention is a kit for a real-time RT-amplification reaction (eg, real-time RT-PCR, real-time RT-LAMP).
  • the kit of the present invention may contain, as the component (b), a component for reverse transcription reaction of RNA strand and a component for real-time amplification reaction.
  • the real-time amplification reaction include an intercalator method and a fluorescent substance labeled probe method.
  • the component (b) for real-time amplification reaction may further include a fluorescent substance or a fluorescent substance-labeled probe for performing such a method as a constituent component.
  • the fluorescent substance include a fluorescent substance used in the intercalator method (eg, SYBR Green I).
  • fluorescent substance-labeled probes include probes in which a fluorescent substance is bound to one of the 5 ′ end or the 3 ′ end and a quencher is bound to the other of the 5 ′ end or the 3 ′ end (for example, TaqMan (registered) Trademark) probe).
  • the kit of the present invention is a kit for real-time RT-PCR.
  • the kit of the present invention may contain, as the component (b), a component for reverse transcription reaction of RNA strand and a component for real-time PCR.
  • the constituents of component (b) include reverse transcriptase, thermostable polymerase, one or more primers, dNTP mixture, reaction buffer, fluorescent substance and fluorescent substance labeled probe as described above, reaction container Is mentioned.
  • the component of the component (b) is not particularly limited as long as it is 1 or more, and may be one or plural (eg, 2, 3, 4, 5, 6). Each component may be provided in a form isolated from each other, for example, stored in different containers (eg, tubes), but may be provided in a premixed form (eg, PreMix) or the like. .
  • the present invention also provides a method for producing a target DNA strand (or a method for synthesizing a target DNA strand).
  • the production method of the present invention includes subjecting an RNA strand to a synthesis reaction of a target DNA strand.
  • the RNA strand As the RNA strand, the RNA strand to be stored in the storage solution of the present invention is used.
  • Examples of the production reaction of the target DNA strand include reverse transcription reaction of RNA strand or RT-amplification reaction.
  • This method may be performed by the kit of the present invention described above.
  • the method for producing the target DNA strand is a method using a real-time RT-amplification reaction (eg, real-time RT-PCR).
  • the RT-amplification reaction may be a one-step RT-amplification reaction (eg, RT-PCR) in which the RT reaction and the amplification reaction are performed simultaneously.
  • the present invention also provides a method for measuring the RNA expression level.
  • the measurement method of the present invention includes using the RNA strand to be stored contained in the storage solution of the present invention as an RNA preparation (ie, control) for measuring the RNA expression level.
  • RNA samples used in the measurement of RNA expression level include mammal-derived RNA samples.
  • mammals include humans, monkeys, cows, pigs, mice, rats, guinea pigs, hamsters, and rabbits. From the viewpoint of clinical application, the mammalian species is preferably human.
  • the RNA sample include a sample (eg, an extract from a cell or tissue) containing an RNA strand (eg, total RNA, mRNA) derived from nature.
  • an RT-amplification reaction can be used. Therefore, in the measurement method of the present invention, for example, (a1 ′) an RNA sample is subjected to an RT-amplification reaction, (b1 ′) an RNA strand to be stored in the storage solution of the present invention is subjected to an RT-amplification reaction. And (c1 ′) a step of verifying the RT-amplification reaction of step (a1 ′) based on the result of step (b1 ′), or a target RNA in the RNA sample based on the result of step (b1 ′) The step of determining the expression level of may be included.
  • Steps (a1 ') and (b1') may be performed separately as described above, or may be performed together.
  • the measurement method of the present invention includes, for example, (a2 ′) a step of combining the RNA sample and the RNA strand to be stored in the storage solution of the present invention, and (b2 ′) RNA in the RNA sample and the storage solution of the present invention.
  • a step of determining the expression level of the target RNA therein may be included.
  • the RNA sample and the measurement method of the present invention may be performed using the kit of the present invention described above.
  • the measurement method of the present invention is a method using a real-time RT-amplification reaction (eg, real-time RT-PCR).
  • the RT-amplification reaction may be a two-step RT-amplification reaction (eg, two-step RT-PCR) in which the RT reaction and the amplification reaction are performed separately, but a one-step RT in which the RT reaction and the amplification reaction are performed simultaneously.
  • -It may be an amplification reaction (eg 1 step RT-PCR).
  • Example 1 Single-stranded RNA preservation test
  • Single-stranded RNA (human TERT gene) to be preserved was prepared by an in vitro transcription method using Ambion (registered trademark) MEGAscript (registered trademark) Kit (manufactured by Applied Biosystems). After purification by RNeasy (registered trademark) mini kit (manufactured by QIAGEN), purity was confirmed using an electrophoresis apparatus MultiNA (manufactured by Shimadzu Corporation). Further, the concentration of the single-stranded RNA after purification was determined by the relationship between the absorbance measured using a spectrophotometer GeneQuant 100 (manufactured by GE Healthcare) and the molecular weight. The single-stranded RNA (human TERT gene) whose purity has been confirmed and concentration has been determined is dissolved in TE buffer to prepare a concentration of 4 ⁇ 10 5 copies / ⁇ L. Obtained.
  • the prepared stock solution is as follows: (1) DEPC water; (2) MS2 RNA aqueous solution (10 ng / ⁇ L); (3) sodium citrate aqueous solution (10 mM); and (4) ammonium aurintricarboxylate aqueous solution (0.25 ⁇ M).
  • MS2 RNA was obtained from Roche Applied Science.
  • MS2 RNA is a single-stranded RNA (RNase free) extracted from phage MS2, has a molecular weight of about 1,200 kDa and is composed of 3569 base.
  • RNase free a single-stranded RNA extracted from phage MS2
  • Each material prepared above was dispensed into a tube, sealed, and stored at 4 ° C. and room temperature (25 ° C., the same applies hereinafter).
  • RNA solutions were collected from each storage tube, and the degree of RNA degradation over time was investigated by RT-PCR.
  • a sample stored at ⁇ 80 ° C. with DEPC water, which has been conventionally used for RNA storage was prepared and measured in the same manner.
  • RT-PCR Details of RT-PCR are as follows. First, 5 ⁇ L was collected from a sample, and reverse transcription reaction was performed using a Transscriptor First Strand cDNA Synthesis Kit (manufactured by Roche Diagnostics) in a total volume of 20 ⁇ L. Then, the solution obtained was diluted 1/5 with herring sperm (10 ng / ⁇ L) and subjected to real-time PCR by an intercalator method using a PCR reactor LightCycler (registered trademark) ST300 (manufactured by Roche Diagnostics). did. The degree of RNA degradation in each solution was investigated from the change over time of the Cp value obtained from this PCR.
  • Transscriptor First Strand cDNA Synthesis Kit manufactured by Roche Diagnostics
  • Tables 1 and 2 show changes over time in the Cp value of single-stranded RNA (human TERT gene) stored at each temperature.
  • the Cp (Crossing Point) value is a value indicating the number of PCR cycles necessary to reach the threshold of the fluorescence intensity meter of the apparatus used (usually set in a range where the intensity increases exponentially). . Maintaining the Cp value over time indicates stable preservation of single-stranded RNA (human TERT gene) (in other words, suppression of degradation of single-stranded RNA). On the other hand, an increase in Cp value with time indicates that single-stranded RNA (human TERT gene) is degraded.
  • PCR amplification curves for single-stranded RNA (human TERT gene) stored at each temperature are shown in FIGS.
  • each material prepared above was subjected to real-time PCR using a hydrolysis probe (fluorescent substance labeled probe).
  • Real-time PCR reaction conditions were the same as those for the real-time PCR except that a hydrolysis probe was used as the detection system.
  • a hydrolysis probe a hydrolysis probe produced in-house by a method well known in the art was used.
  • the degree of RNA degradation in each solution was investigated from the change over time of the Cp value obtained from this PCR.
  • Tables 3 and 4 show changes over time in Cp values of single-stranded RNA (human TERT gene) stored at each temperature.
  • Example 2 Single-stranded RNA preservation test (2) A single-stranded RNA (human TERT gene), which was prepared in the same manner as in Example 1 and whose purity was confirmed and the concentration was determined, was dissolved in TE buffer to prepare 4 ⁇ 10 7 copies / ⁇ L. Thus, the following two storage solutions were obtained. (1) MS2 RNA (10 ng / ⁇ L) aqueous solution (2) Mixed aqueous solution of MS2 RNA (10 ng / ⁇ L) and ammonium aurintricarboxylate (1.0 ⁇ M) The same MS2 RNA as in Example 1 was used.
  • a dilution series (4 ⁇ 10 7 , 4 ⁇ 10 6 , 1 ⁇ 10 7 copies / ⁇ L to 4 ⁇ 10 3 copies / ⁇ L) of single-stranded RNA (human TERT gene) 4 ⁇ 10 5 , 4 ⁇ 10 4 , 4 ⁇ 10 3 copies / ⁇ L) were prepared for each of the two solutions.
  • Each material prepared above was dispensed into tubes, sealed, and stored at 4 ° C. and room temperature.
  • RNA solutions were periodically collected from each storage tube, and the degree of RNA degradation over time was investigated by RT-PCR using the intercalator method. RT-PCR by the intercalator method was performed in the same manner as in Example 1.
  • Tables 5 to 8 show changes with time in Cp values of single-stranded RNA (human TERT gene) stored at each temperature. Further, graphs of the data in Tables 5 to 8 are shown in FIGS.
  • RNA degradation over time was investigated by RT-PCR using a hydrolysis probe (fluorescent substance labeled probe).
  • RT-PCR using a hydrolysis probe was carried out in the same manner as in Example 1.
  • Tables 9 to 12 show changes over time in Cp value of single-stranded RNA (human TERT gene) stored at each temperature. Further, graphs of the data in Tables 9 to 12 are shown in FIGS.
  • Example 3 Total RNA preservation test
  • Total RNA to be preserved was extracted from human-derived cultured cells K562 by a general method and purified. Thereafter, the concentration of total RNA obtained from the absorbance measured using a spectrophotometer GeneQuant 100 (manufactured by GE Healthcare) was determined. The total RNA whose concentration was determined was dissolved in TE buffer and prepared to a concentration of 40 ng / ⁇ L to obtain the following stock solution. The prepared stock solution is as follows. (1) DEPC water (2) MS2 RNA aqueous solution (10 ng / ⁇ L) (3) A sodium citrate aqueous solution (10 mM), (4) Ammonium aurintricarboxylate (0.25 ⁇ M) aqueous solution. The same MS2 RNA as in Example 1 was used.
  • RNA solutions were periodically collected from each storage tube, and the degree of RNA degradation over time was examined by RT-PCR in the same manner as in Example 1.
  • RT-PCR was performed in the same manner as in Example 1 except that human GAPDH was targeted.
  • Tables 13 and 14 show changes over time in the Cp value of the total RNA stored at each temperature. Further, graphs of the data in Tables 13 and 14 are shown in FIGS.
  • RNA stabilizer candidate substance aurintricarboxylic acid has the property of binding to nucleic acid binding sites of nucleic acids and nucleic acid binding proteins, and has been reported to inhibit almost all enzyme reactions against nucleic acids (Thomas Blumenthal et al. Biochemical and Biophysical Research Communications, 55 (3), 680-688 (1973)). However, whether or not RT-PCR (reverse transcription reaction and / or amplification reaction) is inhibited by ATA and, if inhibited, its inhibitory concentration has not been reported.
  • RT-PCR reverse transcription reaction and / or amplification reaction
  • RT-PCR is not inhibited by ATA, or if inhibition is negligible at low concentrations, there is room for further experiments as a stabilizer. Therefore, the influence of ATA on RT-PCR was examined.
  • total RNA extracted and purified from human-derived cultured cells K562 by a general method was used. Thereafter, the total RNA concentration was determined using the absorbance value measured using a spectrophotometer GeneQuant 100 (manufactured by GE Healthcare).
  • RNA concentration 40 ng / ⁇ L (DEPC water is used as a solvent for dissolving the total RNA), ATA (ammonium salt) concentration is 0 mM (no addition), 0.01 mM, 0.1 mM
  • Two-step RT-PCR was performed using the above storage solution without providing a storage period. Specifically, 5 ⁇ L was collected from the stock solution, and reverse transcription reaction was performed in a 20 ⁇ L system using Transscriptor First Strand cDNA Synthesis Kit (manufactured by Roche Diagnostics).
  • the concentration of ATA (ammonium salt) in the PCR solution was ATA (ammonium salt) in the storage solution.
  • ATA ammonium salt
  • Example 5 Investigation of ATA effect on reverse transcription reaction (2) Based on the results of Example 4, it was confirmed to what extent the reverse transcription reaction was inhibited when the ATA (ammonium salt) concentration was lower than 0.01 mM (10 ⁇ M). Using a solution with a total RNA concentration of 40 ng / ⁇ L prepared by the same method as in Example 4, the ATA (ammonium salt) concentration was adjusted to 0 ⁇ M (no addition), 0.1 ⁇ M, and 1.0 ⁇ M 3 A seed stock solution was obtained. Next, reverse transcription reaction and real-time PCR by the intercalator method (that is, two-step RT-PCR) were performed using human WT-1 as a target in the same manner as in Example 4 without providing a storage period.
  • the intercalator method that is, two-step RT-PCR
  • the ATA (ammonium salt) concentration in the reverse transcription reaction solution was 1 of the ATA (ammonium salt) concentration in the storage solution. / 4.
  • the concentration of ATA (ammonium salt) in the PCR solution was ATA (ammonium salt) in the storage solution. ) It was 1/80 of the concentration. As a result, PCR was hardly inhibited when 1.0 ⁇ M and 0.1 ⁇ M ATA (ammonium salt) solutions were used as storage solutions (Table 16).
  • Example 6 Investigation of the effect of ATA on reverse transcription reaction (3) Based on the results of Examples 4 and 5, it was confirmed to what extent the reverse transcription reaction was inhibited at an ATA (ammonium salt) concentration of 2.5 to 7.5 ⁇ M. Using a solution with a total RNA concentration of 40 ng / ⁇ L prepared by the same method as in Example 4, the ATA (ammonium salt) concentration is 0 ⁇ M (no addition), 2.5 ⁇ M, 5.0 ⁇ M, and 7.5 ⁇ M. Four kinds of preservation solutions prepared in the above were obtained.
  • ATA ammonium salt
  • reverse transcription reaction and real-time PCR by the intercalator method were performed using human WT-1 as a target in the same manner as in Example 4 without providing a storage period. Since the volume of the storage solution in the reverse transcription reaction solution (total 20 ⁇ L) was 5 ⁇ L, the ATA (ammonium salt) concentration in the reverse transcription reaction solution was 1 of the ATA (ammonium salt) concentration in the storage solution. / 4. In addition, since the volume of the 1/5 dilution of the reverse transcription reaction solution in the PCR solution (total amount 20 ⁇ L) was 5 ⁇ L, the concentration of ATA (ammonium salt) in the PCR solution was ATA (ammonium salt) in the storage solution.
  • Example 7 Investigation of ATA effect on RT-PCR with hydrolysis probe
  • the optimum concentration of ATA (ammonium salt) that does not inhibit RT-PCR is generally 1.0 ⁇ M. It was confirmed by real-time PCR using the intercalator method. Therefore, in this example, the influence of 1.0 ⁇ M ATA (ammonium salt) on real-time PCR using a hydrolysis probe (fluorescent substance labeled probe) was investigated.
  • a hydrolysis probe produced in-house by a method well known in the art was used.
  • two-step RT-PCR was performed without providing a storage period.
  • the reverse transcription reaction was performed in the same manner as in Example 4.
  • LightCycler registered trademark
  • ST300 manufactured by Roche Diagnostics
  • the ATA (ammonium salt) concentration in the reverse transcription reaction solution was 1 of the ATA (ammonium salt) concentration in the storage solution. / 4.
  • the concentration of ATA (ammonium salt) in the PCR solution was ATA (ammonium salt) in the storage solution. ) It was 1/80 of the concentration. As a result, it was confirmed that RT-PCR with the hydrolysis probe was not inhibited when a 1.0 ⁇ M ATA (ammonium salt) solution was used as a storage solution (Table 18).
  • Example 8 Investigation of the effect of ATA on RT-PCR using single-stranded RNA as a template
  • ATA that does not inhibit RT-PCR by RT-PCR using total RNA as a template
  • the optimum concentration of ammonium salt was 1.0 ⁇ M.
  • the effect of ATA (ammonium salt) on RT-PCR using single-stranded RNA as a template was confirmed.
  • the ATA (ammonium salt) concentration was 0 ⁇ M (no addition), 0.1 ⁇ M
  • Three stock solutions prepared to 1.0 ⁇ M were obtained.
  • reverse transcription reaction and real-time PCR by the intercalator method were performed in the same manner as in Example 4 without providing a storage period. Since the volume of the storage solution in the reverse transcription reaction solution (total amount 20 ⁇ L) was 5 ⁇ L, the ATA (ammonium salt) concentration in the reverse transcription reaction solution was 1 of the ATA (ammonium salt) concentration in the storage solution. / 4. Moreover, since the volume of the 1/5 dilution of the reverse transcription reaction solution in the PCR solution (total amount 20 ⁇ L) was 5 ⁇ L, the concentration of ATA (ammonium salt) in the PCR solution was ATA (ammonium salt) in the storage solution.
  • Example 9 Preservation test of single-stranded RNA by ATA Stability of single-stranded RNA in a solution containing 1.0 ⁇ M ATA (ammonium salt), which was confirmed by previous experiments to not inhibit RT-PCR Sex was tested.
  • ATA ammonium salt
  • a solution of single-stranded RNA (human TERT gene) concentration 4 ⁇ 10 5 copies / ⁇ L prepared by the same method as in Example 1 ATA (ammonium salt) concentration was 0 ⁇ M (no addition), 1.0 ⁇ M
  • Two kinds of stock solutions prepared so as to be obtained were obtained. Each material prepared above was dispensed into tubes, sealed, and stored at 4 ° C. and room temperature.
  • RNA solutions were collected from each tube, and the degree of RNA degradation over time was examined by RT-PCR.
  • RT-PCR reverse transcription reaction and real-time PCR by the intercalator method (ie, 2-step RT-PCR) were performed in the same manner as in Example 4.
  • Tables 20 and 21 show changes over time in the Cp value of single-stranded RNA (human TERT gene) stored at each temperature.
  • FIG. 13 shows the PCR amplification curve for single-stranded RNA (human TERT gene) stored at 4 ° C. and room temperature for 2 weeks.
  • Example 10 Preservation test of single-stranded RNA by ATA
  • a solution containing only ribosomal RNA single-stranded RNA capable of stabilizing the RNA strand
  • both ribosomal RNA and ATA ammonium salt
  • the stability of single-stranded RNA was tested.
  • a single-stranded RNA human TERT gene
  • concentration was determined by the same method as in Example 1 was dissolved in TE buffer to prepare a concentration of 4 ⁇ 10 5 copies / ⁇ L.
  • the following stock solution was obtained.
  • the prepared stock solution is as follows: (1) DEPC water; (2) An aqueous solution containing ribosomal RNA (10 ng / ⁇ L); and (3) an aqueous solution containing ribosomal RNA (10 ng / ⁇ L) and ATA (ammonium salt) (1.0 ⁇ M).
  • Ribosomal RNA (16s, 23s) was obtained from Roche Applied Science. This ribosomal RNA (16s, 23s) is E. coli. E. coli MRE600-derived single-stranded RNA (RNase free), which is composed of 1500 base (16 s) and 2900 base (23 s). Each material prepared above was dispensed into tubes, sealed, and stored at 4 ° C., room temperature, and 37 ° C. After 1 week and 2 weeks, RNA solutions were collected from each tube, and the degree of RNA degradation over time was examined by RT-PCR. For RT-PCR, reverse transcription reaction and real-time PCR by the intercalator method (ie, two-step RT-PCR) were performed in the same manner as in Example 4.
  • RNase free RNase free
  • Tables 22 to 24 show changes over time in Cp values of single-stranded RNA (human TERT gene) stored at each temperature.
  • ribosomal RNA suppressed the degradation of single-stranded RNA (human TERT gene).
  • phage RNA MS2 RNA was superior to ribosomal RNA (16s, 23s) in stabilizing RNA strands. .
  • the present invention is useful for stabilizing RNA strands. Therefore, the present invention provides a method of utilizing an RNA strand, for example, in a synthesis reaction of a target DNA strand from an RNA strand (eg, reverse transcription reaction, RT-PCR), or in a synthesis reaction of a target DNA strand from an RNA strand. Useful as reagents and kits.

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Abstract

La présente invention concerne un procédé de stabilisation de brin d'ARN qui permet de fournir un échantillon d'ARN pour mettre en œuvre une réaction comme la PCR en temps réel. La présente invention concerne spécifiquement : une solution de conservation de brin d'ARN comprenant un brin d'ARN à conserver et un ARN simple brin pouvant stabiliser un brin d'ARN (et/ou 0,1 à 2,5 µM d'un sel d'acide aurinetricarboxylique) ; un procédé de production d'une solution de brin d'ARN ; un procédé de stabilisation d'un brin d'ARN ; un kit contenant une solution de brin d'ARN ; un procédé de production d'un brin d'ADN à partir d'un brin d'ARN conservé dans une solution de conservation de brin d'ARN ; un procédé de mesure du niveau d'expression de l'ARN à l'aide d'un brin d'ARN conservé dans une solution de conservation de brin d'ARN au titre d'échantillon d'ARN dont le niveau d'expression doit être mesuré ; etc.
PCT/JP2011/064366 2010-06-25 2011-06-23 Solution de conservation de brin d'arn WO2011162326A1 (fr)

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WO1990000198A1 (fr) * 1988-06-27 1990-01-11 Health Research, Inc. Vecteur de stabilisation d'arn
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