JP7824596B2 - Method for suppressing non-specific nucleic acid amplification - Google Patents

Description

The present invention relates to a method for suppressing nonspecific nucleic acid amplification in an RT-RamDA reaction.

The RT-RamDA method is an amplification reverse transcription method that uses RNA as a template to amplify cDNA by incubating a mixture containing template RNA, primers, DNA strand-specific RNA:DNA hybrid strand degrading enzyme, RNase H minus reverse transcriptase, and a substrate (Patent Document 1). It has been reported that the RT-RamDA method can increase cDNA by 10 to 100 times compared to conventional reverse transcription reactions. This makes it possible to detect low-expression genes from minute amounts of RNA, which was previously difficult, and to expand the number of detectable genes, making it a promising nucleic acid amplification technology.

International Publication No. 2016/052619

The inventors have discovered that nonspecific nucleic acid amplification sometimes occurs during the RT-RamDA reaction, resulting in smearing on the high molecular weight side when detected by electrophoresis. This has led to the development of a method for suppressing such unintended nonspecific nucleic acid amplification. While not wishing to be bound by theory, one possible cause of this nonspecific amplification is inappropriate annealing of primers or the formation of aggregates of abnormal amplification products. Therefore, one object of the present invention is to provide a means for suppressing the above-mentioned nonspecific nucleic acid amplification that occurs in the RT-RamDA method.

As a result of extensive research to achieve the above-mentioned objective, the inventors discovered that in the RT-RamDA method, nonspecific nucleic acid amplification reactions can be suppressed by performing the RT-RamDA reaction in the presence of template RNA and a chaotropic agent in the RT-RamDA reaction solution, leading to the completion of the present invention.

That is, the present invention includes the following aspects.
[Item 1] A method for suppressing non-specific nucleic acid amplification in an RT-RamDA reaction, characterized by allowing a template RNA and a chaotropic agent to coexist in an RT-RamDA reaction solution.
[Item 2] The method according to Item 1, wherein a template RNA-containing biological sample to be subjected to an RT-RamDA reaction is treated with a cell lysis agent containing a chaotropic agent to prepare a template RNA-containing biological sample solution containing a chaotropic agent, and the template RNA-containing biological sample solution containing the chaotropic agent is mixed with an RT-RamDA reaction solution, thereby allowing the template RNA and the chaotropic agent to coexist in the RT-RamDA reaction solution.
[Item 3] The method according to Item 1 or 2, wherein the final concentration of the chaotropic agent in the RT-RamDA reaction solution is more than 0 mM and not more than 50 mM.
[Item 4] The method according to any one of Items 1 to 3, wherein the final concentration of the chaotropic agent in the RT-RamDA reaction solution is more than 0 mM and 20 mM or less.
[Item 5] The method according to any one of Items 1 to 4, wherein the chaotropic agent is at least one chaotropic agent selected from the group consisting of guanidinium ions, urea ions, iodide ions, lithium ions, and salts thereof.
[Item 6] The method according to any one of Items 1 to 5, wherein the chaotropic agent is a guanidinium salt.
[Item 7] The method according to any one of Items 1 to 6, wherein the chaotropic agent is guanidine thiocyanate.
[Item 8] The method according to any one of Items 1 to 7, wherein the RT-RamDA reaction solution further contains an inorganic salt.
[Item 9] The method according to Item 8, wherein the inorganic salt is at least one selected from the group consisting of potassium salts, manganese salts, and magnesium salts.
[Item 10] A method for stabilizing an RT-RamDA reaction, characterized by allowing a template RNA and a chaotropic agent to coexist in an RT-RamDA reaction solution.
[Item 11] A kit for use in the method according to any one of Items 1 to 10, comprising an RT-RamDA reaction solution containing a chaotropic agent.
[Item 12] A kit for use in the method according to any one of Items 1 to 10, comprising a cell lysis solution containing a chaotropic agent.

The present invention effectively suppresses nonspecific nucleic acid amplification reactions in RT-RamDA reactions. This makes it possible to perform highly reproducible and stable RT-RamDA reactions without compromising the quantitative performance of the RT-RamDA method.

FIG. 1 shows the amplification curves of quantitative PCR under conditions 1 to 4 in Example 1.

The following is a detailed description of an embodiment of the present invention, but the present invention is not limited thereto. All non-patent documents and patent documents described in this specification are incorporated herein by reference. In addition, the term "~" in this specification means "greater than or equal to, less than or equal to." For example, "X to Y" in the specification means "greater than or equal to X and less than or equal to Y." In addition, "and/or" in this specification means either one or both.

In one embodiment, the method of the present invention for suppressing nonspecific nucleic acid amplification in an RT-RamDA reaction is characterized in that, when performing the RT-RamDA method, the RT-RamDA reaction is carried out in the presence of template RNA and a chaotropic agent in the RT-RamDA reaction solution. By amplifying cDNA from template RNA in the presence of a chaotropic agent in the RT-RamDA reaction solution, nonspecific nucleic acid amplification that may occur in the RT-RamDA reaction can be effectively suppressed or reduced. Furthermore, the method of the present invention can also be considered a method for stabilizing the RT-RamDA reaction, given that it suppresses nonspecific nucleic acid amplification and enables the RT-RamDA reaction to be carried out stably and with good reproducibility.

・RT-RamDA method (amplified reverse transcription method in which reverse transcription of template RNA amplifies cDNA using RNA as a template)
The RT-RamDA method is a method for amplifying nucleic acids, which includes a step of incubating a mixture containing template RNA, primers, DNA strand-specific RNA:DNA hybrid strand degrading enzyme, RNase H minus reverse transcriptase, and a substrate. Therefore, the RT-RamDA reaction solution of the present invention contains at least the above-mentioned components (i.e., template RNA, primers, DNA strand-specific RNA:DNA hybrid strand degrading enzyme, RNase H minus reverse transcriptase, and substrate). In the RT-RamDA method, a complementary strand DNA (cDNA) of a template RNA is synthesized using the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase, and the cDNA strand of the RNA-cDNA hybrid strand is randomly cleaved by a DNA strand-specific RNA:DNA hybrid strand degrading enzyme. Starting from the cleavage site, the 3'-side cDNA strand is stripped from the RNA by the strand displacement activity of the RNase H minus reverse transcriptase, and a new cDNA strand is synthesized at the site stripped by the RNase H minus reverse transcriptase. Details of the RT-RamDA method are described in U.S. Patent Application Publication No. 2017/0275685 (the entire contents of which are incorporated herein by reference), etc.

In one embodiment, the present invention is characterized by suppressing nonspecific nucleic acid amplification that may occur during the RT-RamDA method. As used herein, "nonspecific nucleic acid amplification" is not particularly limited and refers to the generation of amplification products other than normal specific amplification products during the RT-RamDA method. Examples of nonspecific amplification product formation include the formation of linear multimers (concatemers) of amplification products, the formation of aggregates of linear multimers (concatemers) of amplification products, the formation of linear multimers (concatemers) between primers, the formation of aggregates of linear multimers (concatemers) between primers, the formation of mismatch annealing of primers, and/or the formation of primer dimers. The present invention does not necessarily suppress the formation of all of the nonspecific amplification products listed above, but may suppress, for example, the formation of at least one of the nonspecific amplification products listed above.
In this specification, the criteria for determining whether nonspecific nucleic acid amplification is suppressed include, for example, whether the linear relationship between the initial template amount and the Ct value is improved and/or the PCR efficiency is improved (e.g., the PCR efficiency is closer to 100%) when a chaotropic agent is coexisted in the RT-RamDA reaction solution compared to when a chaotropic agent is not used.

Chaotropic Agent: One of the features of the present invention is the presence of a chaotropic agent in the RT-RamDA reaction solution. A chaotropic agent is a component that inhibits the stabilization of intermolecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects. Therefore, chaotropic agents can disrupt the three-dimensional structure of macromolecules such as proteins, DNA, or RNA, denaturing them. Therefore, there is concern that the use of an excessive amount of chaotropic agent may affect the RT-RamDA reaction. However, without being bound by theory, the inventors' studies suggest that the presence of a chaotropic agent in the RT-RamDA reaction solution under given conditions can exert effects such as preventing primer misannealing and/or appropriately dissociating aggregates of abnormal amplification products, thereby suppressing unintended nonspecific nucleic acid amplification and stabilizing the RT-RamDA reaction.

The chaotropic agent that can be used in the present invention is not particularly limited as long as it achieves the effects of the present invention, but can include, for example, at least one selected from the group consisting of guanidinium ions, urea ions, iodide ions, lithium ions, and salts thereof (e.g., hydrochlorides, thiocyanates, perchlorates, etc.). From the standpoint of relatively small inhibitory effects on the RT-RamDA reaction and high suppression of nonspecific nucleic acid amplification in the RT-RamDA reaction, it is preferable to use guanidinium ions and/or salts thereof as the chaotropic agent, more preferably guanidinium salts, and even more preferably guanidine thiocyanate and/or guanidine hydrochloride, with guanidine thiocyanate being particularly preferred.

The amount of chaotropic agent added to the RT-RamDA reaction solution is not particularly limited as long as the effects of the present invention are achieved. However, as mentioned above, if an excessive amount of chaotropic agent is present in the RT-RamDA reaction solution, there is a concern that the RT-RamDA reaction may be more easily inhibited. Therefore, the concentration of the chaotropic agent used in the present invention, as a final concentration in the RT-RamDA reaction solution, is preferably, for example, 50 mM or less, more preferably 40 mM or less, even more preferably 30 mM or less, and particularly preferably 20 mM or less. The lower limit of the final concentration of the chaotropic agent in the RT-RamDA reaction solution is not particularly limited as long as the effects of the present invention are achieved. However, for example, it can be an amount greater than 0 mM, preferably 1 mM or more, more preferably 3 mM or more, even more preferably 5 mM or more, even more preferably 7 mM or more, and particularly preferably 10 mM or more.

Template RNA
The template RNA used in the RT-RamDA reaction can be any RNA, such as RNA extracted from tissues or cells, or RNA extracted from tissues or cells and then further purified (e.g., purified RNA treated by any purification method known in the art, such as ethanol precipitation or column purification). Conveniently, a biological sample (e.g., cells or tissue) to be analyzed by the RT-RamDA method can be lysed with a cell lysing agent (herein also referred to as a "template RNA-containing biological sample") and used as is in the RT-RamDA reaction without further extraction or purification steps. The amount of template RNA contained in the RT-RamDA reaction solution is not particularly limited as long as the effects of the present invention are achieved. However, from the viewpoints of more reliably and accurately performing the RT-RamDA reaction and easily obtaining stable, highly reproducible results, the amount is preferably 0.1 pg/μl to 10 ng/μl, more preferably 0.1 pg/μl to 1 ng/μl. In the present invention, when a "template RNA-containing biological sample" is used as the template RNA, the biological sample may be used in an amount such that the amount of template RNA contained in the biological sample falls within the above concentration range.

The type of cell or tissue from which template RNA to be amplified is extracted is not particularly limited, and any type of cell or tissue may be used. For example, the number of cells can be appropriately adjusted using a cell sorter. For example, RNA extracted from a small number of cells (e.g., 1 to 100, preferably 1 to 10, more preferably 1 or 2, more preferably 1) can also be used.
Methods for extracting RNA from cells typically include a step of lysing the cells using a cell lysis agent containing a cell lysis component.
The cell lysis agent may, for example, contain a surfactant as a cell lysis component. Examples of surfactants include, but are not limited to, anionic surfactants (e.g., sodium dodecyl sulfate, sodium cholate, sodium deoxycholate), cationic surfactants (e.g., cetyltrimethylammonium bromide), nonionic surfactants (e.g., octylphenol ethoxylate, polyoxyethylene lauryl ether, polyoxyethylene stearyl ether, polyoxyethylene sorbitan monolaurate), and zwitterionic surfactants (e.g., 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid). Furthermore, the cell lysis agent may optionally contain, as a cell lysis component, a protease (e.g., protease K), an RNase inhibitor, or a combination of two or more thereof. In further specific embodiments, the cell lysis agent used in the present invention may contain a chaotropic agent (e.g., urea, lithium perchlorate, guanidinium salts such as guanidine hydrochloride, etc.). In other specific embodiments, the cell lysis agent may be chaotropic-free. The cell lysis agent is usually used in the form of an aqueous solution in which the cell lysis component as described above is dissolved in an aqueous solvent such as water (preferably nuclease-free water).

In certain embodiments, the RT-RamDA method of the present invention can use a template RNA-containing biological sample obtained by lysing a biological sample (e.g., cells, tissues, etc.) using a cell lysis agent containing a chaotropic agent. A template RNA-containing biological sample obtained by treatment with such a cell lysis agent containing a chaotropic agent will directly become a biological sample solution containing the chaotropic agent unless further extraction and purification steps are performed. Therefore, when mixed with an RT-RamDA reaction solution, the chaotropic agent will be carried over into the RT-RamDA reaction solution. When such a biological sample solution containing a chaotropic agent is used as a template RNA-containing biological sample, there is an advantage in that there is no need to separately add a chaotropic agent to the RT-RamDA reaction solution.

When using a biological sample solution obtained by lysing a biological sample with a cell lysis agent containing a chaotropic agent as a template RNA-containing biological sample, it is preferable to use a cell lysis solution in which the total final concentration of the chaotropic agent in the RT-RamDA reaction solution is preferably 50 mM or less, more preferably 40 mM or less, even more preferably 30 mM or less, and particularly preferably 20 mM or less, regardless of whether a chaotropic agent is added separately. Similarly, it is preferable to use a cell lysis solution in which the lower limit of the total final concentration of the chaotropic agent in the RT-RamDA reaction solution is adjusted to, for example, more than 0 mM, preferably 1 mM or more, more preferably 3 mM or more, even more preferably 5 mM or more, even more preferably 7 mM or more, and especially 10 mM or more.

DNA strand-specific RNA:DNA hybrid strand-degrading enzyme : The DNA strand-specific RNA:DNA hybrid strand-degrading enzyme is preferably an enzyme having the activity of cleaving the DNA strand in an RNA-DNA hybrid strand. Examples of such enzymes that can be used include double-strand-specific DNA degrading enzymes and non-specific DNA degrading enzymes. The amount of DNA strand-specific RNA:DNA hybrid strand-degrading enzyme contained in the RT-RamDA reaction solution is not particularly limited as long as the effects of the present invention are achieved. However, from the viewpoints of enabling the RT-RamDA reaction to be carried out more reliably and accurately and facilitating the stable production of highly reproducible results, the amount is preferably 0.01 U/μl to 0.1 U/μl.

The double-strand-specific degrading enzyme (also referred to as double-strand-specific nuclease; DSN) may be an enzyme derived from a prokaryote or a eukaryote, but preferably, a double-strand-specific DNA degrading enzyme derived from a crustacean or a modified form thereof can be used. Specific examples include the following:
・ Solenocera melanthho (shrimp shrimp) DNase
・ Penaeus japonicus (prawn) DNase
・ Paralithodes camtschaticus (king crab) DSN
・Pandalus borealis (sea shrimp) dsDNase
・ Chionoecetes opilio (snow crab) DSN
Other DSN homologs The double-strand-specific DNase is preferably an enzyme that has DNA degradation activity even at temperatures below 60° C. Among the above, shrimp-derived double-strand-specific DNase or a modified form thereof is preferred.
Commercially available double-strand-specific DNA degrading enzymes can be used, including dsDNase (ArcticZymes), H1-dsDNase (ArcticZymes), dsDNase (Thermo Scientific), Shrimp DNase, Recombinant (Affymetrix), Atlantis dsDNase (Zymo Research), and Thermolabile Nuclease (Roche).

Examples of nonspecific DNA degrading enzymes include enzymes that have the activity to cleave the DNA strand of an RNA-DNA hybrid chain, but have substantially no activity to cleave the RNA strand of an RNA-DNA hybrid chain or single-stranded RNA, and preferably enzymes whose activity to cleave single-stranded DNA is lower than the activity to cleave the DNA strand of an RNA-DNA hybrid chain. Nonspecific DNA degrading enzymes are preferably enzymes that have DNA degradation activity even at temperatures below 60°C. Commercially available nonspecific DNA degrading enzymes such as DNase I (manufactured by Thermo Fisher) can also be used.
The non-specific DNA degrading enzyme that can be used may be an enzyme derived from a prokaryote or a eukaryote, but preferably, a non-specific DNA degrading enzyme derived from a mammal or a variant thereof, more preferably, a non-specific DNA degrading enzyme derived from a bovine or a variant thereof.

The above-mentioned variant refers to an enzyme obtained by modifying a naturally occurring amino acid sequence. Specifically, it refers to an enzyme consisting of an amino acid sequence that has 80% or more (preferably 90% or more, more preferably 95% or more) sequence identity with a naturally occurring amino acid sequence, as well as an enzyme consisting of an amino acid sequence in which one or several (e.g., 1 to 10, preferably 1 to 5, more preferably 1 to 3) amino acids have been deleted, substituted, and/or added to the naturally occurring amino acid sequence.

The single-stranded DNA binding protein RT-RamDA reaction solution may further contain a single-stranded DNA binding protein. A single-stranded DNA binding protein is typically used together with a DNA-strand-specific RNA:DNA hybrid strand degrading enzyme. In particular, when a non-specific DNA degrading enzyme is used as the DNA-strand-specific RNA:DNA hybrid strand degrading enzyme, it is preferable to include a single-stranded DNA binding protein. Examples of single-stranded DNA binding proteins include T4 gene 32 protein, RecA, SSB (single-stranded DNA binding protein), and combinations of two or more of these. When the RT-RamDA reaction solution contains a single-stranded DNA binding protein, the amount of the single-stranded DNA binding protein added is not particularly limited as long as the effects of the present invention are achieved. However, from the viewpoints of more reliably carrying out the RT-RamDA reaction and easily obtaining stable, highly reproducible results, the amount is preferably 10 ng/μl to 100 ng/μl.

Primers used in the RT-RamDA reaction include primers specific to the template RNA, oligo dT primers, random primers, and combinations of two or more of these. When oligo dT primers and random primers are used in combination, the molar ratio of oligo dT primers to random primers can be, for example, 1:5 to 1:15, preferably 1:8 to 1:12.
Examples of random primers include completely random primers and NSR (Not So Random) primers.

A completely random primer is a mixture of primers with various base sequences, each of which is a completely random base sequence. A completely random primer may contain a sequence that is completely identical (or completely complementary) to an rRNA sequence. Examples of completely random primers include completely random pentamers, completely random hexamers, completely random heptamers, completely random octamers, and combinations of these. For example, a completely random hexamer may be a mixture of all possible base sequences (46 types) using the four types of nucleotides (A, T, C, G).

NSR primers are fully random primers that have been stripped of primers that have sequences completely complementary to rRNA sequences, such as 18S rRNA sequences, 28S rRNA sequences, 12S rRNA sequences, 16S rRNA sequences, and combinations thereof.
Examples of NSR primers include fully random hexamers obtained by excluding hexamers having sequences completely complementary to rRNA sequences. Furthermore, NSR primers can also be obtained by excluding those having sequences completely complementary to rRNA sequences from primer sets such as fully random pentamers, fully random heptamers, and fully random octamers.
By using such NSR primers, mRNA and the like can be analyzed with higher sensitivity.

For details of NSR primers, see 1) Amour et al., Digital transcriptome profiling using selective hexamer priming for cDNA synthesis, Nature Methods, Vol. 6, No. 9, 2009, pp. 647-649, and 2) Ozsolak et al., Digital transcriptome profiling from attomole-level RNA samples, Genome Research, Vol. 20, 2010, pp. 519-525, 3) and the like, as described in U.S. Patent Application Publication No. 2010/0029511 (the entirety of which is incorporated herein by reference).
The length of the primer is, for example, 5 bases or more, preferably 6 bases or more, from the viewpoint of annealing, and, for example, 30 bases or less, preferably 25 bases or less, more preferably 20 bases or less, from the viewpoint of synthesis.
The concentration of the primer in the RT-RamDA reaction solution is not particularly limited, but is, for example, 1 to 10 μM, preferably 2 to 6 μM, and more preferably 3 to 5 μM.

- Deoxyribonucleotide (substrate)
As deoxyribonucleotide, deoxyribonucleoside triphosphate is preferred.As deoxyribonucleotide triphosphate, for example, deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxyuridine triphosphate (dUTP), their derivatives, and the combination of two or more of them.Among these, the mixture of dCTP, dGTP, dATP and dTTP, the mixture of dCTP, dGTP, dATP and dUTP, the mixture of dCTP, dGTP, dATP, dTTP and dUTP, etc. are preferred. The amount of deoxyribonucleotide (substrate) contained in the RT-RamDA reaction solution is not particularly limited as long as it achieves the effects of the present invention, but it can preferably be set to 0.1 to 5 mM from the viewpoint of enabling the RT-RamDA reaction to be carried out more reliably and making it easier to obtain stable and highly reproducible results.

RNase H minus reverse transcriptase: The RNase H minus reverse transcriptase used in the RT-RamDA reaction refers to any protein (enzyme) that has reverse transcription activity (RNA-dependent DNA polymerase activity) but does not have RNase H activity. Examples of RNase H minus reverse transcriptases include avian myeloblastosis virus reverse transcriptase (AMV-RT), Moloney murine leukemia virus reverse transcriptase (MMLV-RT), human immunovirus reverse transcriptase (HIV-RT), EIRV-RT, RAV2-RT, C. Examples of suitable reverse transcriptases include C. hydrogenogormans DNA polymerase, rTth DNA polymerase, SuperScript I, SuperScript II, mutants thereof, and derivatives thereof. Of these, MMLV-RT is preferred. The amount of RNase H minus reverse transcriptase contained in the RT-RamDA reaction solution is not particularly limited as long as the effects of the present invention are achieved. However, from the viewpoints of more reliably carrying out the RT-RamDA reaction and easily obtaining stable, highly reproducible results, the amount is preferably 0.2 to 2 U/μl.

The RT-RamDA reaction solution of the present invention may be provided as a reagent containing RNase H minus reverse transcriptase from the beginning, or may be prepared immediately before use by adding it to the RT-RamDA reaction solution before the RT-RamDA reaction. When added to the RT-RamDA reaction solution immediately after use, the RNase H minus reverse transcriptase used may be in its original state, lyophilized, or diluted with water (preferably nuclease-free water). For example, the RT-RamDA reaction solution may be diluted to, for example, 1/20 to 1/30, so that the final concentration of the RNase H minus reverse transcriptase in the RT-RamDA reaction solution falls within the above-mentioned range.

Inorganic Salt : The RT-RamDA reaction solution of the present invention preferably further contains an inorganic salt. The presence of an inorganic salt in the RT-RamDA reaction solution allows the RT-RamDA reaction to proceed more efficiently. The type of inorganic salt that can be used in the present invention is not particularly limited as long as the effects of the present invention are achieved, but examples include potassium salts, manganese salts, and/or magnesium salts, and preferably potassium salts. When an inorganic salt is present in the RT-RamDA reaction solution, the final concentration of the inorganic salt in the reaction solution is not particularly limited, but can be, for example, 20 to 100 mM.

DNA polymerase The RT-RamDA reaction solution of the present invention may or may not contain a DNA polymerase. When the RT-RamDA reaction solution of the present invention contains a DNA polymerase, any DNA polymerase can be used, including, but not limited to, the following DNA polymerases:
Taq, Tbr, Tfl, Tru, Tth, Tli, Tac, Tne, Tma, Tih, Tfi, Pfu, Pwo, Kod, Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENT™, DEEPVENT™, and variants thereof

The RT-RamDA reaction solution of the present invention may contain an RNase inhibitor . The RNase inhibitor is not particularly limited, and examples thereof include proteins derived from human placenta, rat lung, or pig liver.

The RT-RamDA reaction solution of the present invention may further contain other additives, such as buffers, salts, and combinations of two or more thereof.
Examples of buffers include Tris, Tricine, Bis-Tricine, Hepes, Mops, Tes, Taps, Pipes, Caps, and combinations of two or more of these. Buffers are usually dissolved in water (preferably nuclease-free water) and used in the form of an aqueous solution.
Examples of salts include chlorides (e.g., lithium chloride, sodium chloride, potassium chloride, magnesium chloride, manganese chloride), acetates (e.g., lithium acetate, sodium acetate, potassium acetate, magnesium acetate, manganese acetate), sulfates (e.g., potassium sulfate, magnesium sulfate, manganese sulfate), and combinations of two or more thereof.

In the incubation RT-RamDA reaction, a nucleic acid amplification reaction is carried out by incubating an RT-RamDA reaction solution containing the template RNA to be analyzed, primers, DNA strand-specific RNA:DNA hybridization enzyme, RNase H minus reverse transcriptase, and substrate, as well as single-stranded DNA-binding protein and other optional components, under specified conditions. Incubation in the RT-RamDA reaction may be carried out under isothermal conditions or thermal cycling conditions. The reverse transcriptase may then be inactivated as needed. The method for inactivating reverse transcription is not particularly limited, but may include, for example, incubation at 90 to 100°C for a specified time (e.g., 1 to 10 minutes).

(1) Isothermal Conditions When incubation is performed under isothermal conditions, it can be performed at a predetermined temperature between 25°C and less than 50°C, preferably between 30 and 45°C, more preferably between 35 and 40°C, for example, at 37°C for a predetermined time (for example, 5 to 180 minutes, preferably 10 to 150 minutes).
Incubation at a predetermined temperature between 25°C and less than 50°C may be carried out in two or more stages. For example, incubation may be performed at a predetermined temperature between 25°C and less than 30°C for 5 to 15 minutes, then at a predetermined temperature between 30°C and less than 35°C for 5 to 15 minutes, and then at a predetermined temperature between 35°C and less than 50°C for a predetermined time (for example, 5 to 60 minutes).
After incubation at a predetermined temperature between 25° C. and less than 50° C., the mixture may be incubated at a predetermined temperature between 50° C. and less than 100° C., for example. The incubation at a predetermined temperature between 50° C. and less than 100° C. may be carried out in two or more stages. For example, the mixture may be incubated at a predetermined temperature between 50° C. and less than 80° C. for 5 to 15 minutes, and then at a predetermined temperature between 80 and 90° C. for 5 to 15 minutes.

(2) Thermal Cycle Conditions When incubation is performed under thermal cycle conditions, for example, a predetermined temperature T1 (e.g., 25°C) between 20°C and less than 30°C and a predetermined temperature T2 (e.g., 37°C) between 30 and 45°C are combined, and a predetermined time at T1 (e.g., 1 to 3 minutes, e.g., 2 minutes) and a predetermined time at T2 (e.g., 1 to 3 minutes, e.g., 2 minutes) are defined as one cycle, and this cycle may be repeated preferably 10 to 40 cycles, more preferably 15 to 35 cycles. Note that prior to the above thermal cycle, for example, incubation may be performed at a predetermined temperature between 25°C and less than 30°C for a predetermined time (e.g., 5 to 15 minutes), then at a predetermined temperature between 30°C and less than 35°C for a predetermined time (e.g., 5 to 15 minutes), and then at a predetermined temperature between 35°C and less than 50°C for a predetermined time (e.g., 1 to 5 minutes). After the thermal cycle, the mixture may be incubated at a predetermined temperature between 50°C and less than 80°C for a predetermined time (e.g., 5 to 15 minutes), and then at a predetermined temperature between 80 and 90°C for a predetermined time (e.g., 5 to 15 minutes).

The nucleic acid amplification method according to the present invention can be used as part of an RT-PCR method or an RT-qPCR method using a trace amount of RNA (for example, a trace amount of RNA equivalent to one cell to several hundred cells).
The nucleic acid amplification method according to the present invention can be used in RNA sequencing using minute amounts of RNA (for example, minute amounts of RNA equivalent to one cell to several hundred cells).

When PCR or qPCR is performed after the RT-RamDA reaction is completed, the PCR reaction solution or qPCR reaction solution may contain the components of the RT-RamDA reaction solution (e.g., enzymes such as DNA strand-specific RNA:DNA hybridization enzymes and RNase H minus reverse transcriptase) as they are, or may be inactivated components. These PCR reaction solutions or qPCR reaction solutions may contain components for DNA amplification reactions (e.g., primers, deoxyribonucleotides, and DNA polymerase), or if the RT-RamDA reaction solution contains components necessary for DNA amplification reactions, such as DNA polymerase, the RT-RamDA reaction solution can be used as a PCR reaction solution or qPCR reaction solution as is, and the two compositions can be referred to interchangeably.
Primers used for DNA amplification in PCR reaction solutions or qPCR reaction solutions are preferably primers (forward primers, reverse primers) specific to DNA (including DNA reverse transcribed from RNA).
Furthermore, a composition for DNA amplification, such as a PCR reaction solution or a qPCR reaction solution, may contain an anti-DNA polymerase antibody, a reaction buffer, a metal ion (such as a magnesium ion), a fluorescent dye, a fluorescently labeled probe, or a combination of two or more of these.

When DNA amplification in a PCR reaction, qPCR reaction, or the like is carried out under thermal cycling conditions, incubation under the thermal cycling conditions may be performed for a cycle consisting of, for example, a predetermined temperature between 80°C and less than 100°C for a predetermined time (e.g., 10 to 30 seconds) and a predetermined temperature between 50°C and 70°C for a predetermined time (e.g., 30 seconds to 2 minutes), with this cycle being repeated preferably 10 to 50 cycles, and more preferably 15 to 40 cycles, but is not limited to these.

The present invention further relates to a kit capable of suppressing nonspecific nucleic acid amplification, which can be used in the above-described method of the present invention. Such a kit of the present invention can be provided as an RNA analysis kit, etc., which suppresses nonspecific nucleic acid amplification and enables stable and accurate RT-RamDA reactions. The kit of the present invention as described above is not particularly limited, as long as it is configured to allow template RNA and a chaotropic agent to coexist in the RT-RamDA reaction solution. For example, the kit of the present invention can be a kit comprising an RT-RamDA reaction solution containing a chaotropic agent (e.g., a premixed RT-RamDA reaction reagent containing a chaotropic agent) and/or a cell lysis solution containing a chaotropic agent (e.g., a premixed cell lysis reagent containing a chaotropic agent). That is, the chaotropic agent can be contained in either or both of the RT-RamDA reaction reagent and the cell lysis reagent, but preferably is adjusted so that the total amount of chaotropic agent in the RT-RamDA reaction solution after mixing with the template RNA is greater than 0 mM and less than or equal to 50 mM in final concentration. The kit of the present invention may further contain other reagents, buffers, etc. necessary for carrying out the nucleic acid amplification reaction, as desired. Examples of other reagents include primers and deoxyribonucleotide triphosphates. The RT-RamDA reaction solution, and the cell lysis solution and other reagents that may be added as needed, may be provided in a single container, or may be provided with each reagent contained in a separate container.

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

Example 1: Inhibition of nonspecific nucleic acid amplification by guanidine thiocyanate in the RT-RamDA method (purified RNA 5 pg to 10 ng)
In this example, the following test was carried out to confirm the influence of a chaotropic agent on non-specific nucleic acid amplification in the RT-RamDA method.
Guanidine thiocyanate was added to the sample solution (RT-RamDA reaction solution), and single-stranded cDNA was synthesized by the RT-RamDA reaction. Subsequently, quantitative polymerase chain reaction (qPCR) was used to compare the amplification curves with and without guanidine thiocyanate. Specifically, this was performed using the following method.
The nucleic acid fragment sample used in this example was 5 pg to 10 ng of RNA purified from NIH3T3 cells using an RNeasy Mini Kit (Qiagen).
To reverse transcribe 5 pg to 10 ng of this RNA by the RT-RamDA method, the components contained in the sample solution (RT-RamDA reaction solution) used in this example and their final concentrations in the sample solution are shown in Table 1 below.

The RT-RamDA method was carried out using 10 μl of a sample solution (RT-RamDA reaction solution) prepared by adding purified RNA to the above composition. The reaction was carried out under the following isothermal conditions: 25°C for 10 minutes, 30°C for 10 minutes, 37°C for 30 minutes, 50°C for 5 minutes, and 95°C for 5 minutes.
Then, the DNA amounts were compared using the following quantitative polymerase chain reaction (qPCR).
Specifically, the RT-RamDA reaction was performed in the aforementioned sample solution (RT-RamDA reaction solution), followed by a 5-fold dilution with nuclease-free water (Qiagen), and 2 μl of the diluted sample solution was used in the qPCR reaction. qPCR after this RT-RamDA reaction was performed using StepOne Plus (Life Technologies) under the following conditions:
The qPCR reaction solution [20 μl (THUNDERBIRD™ SYBR qPCR Mix (TOYOBO Corporation), 6 pmol forward primer, 6 pmol reverse primer, 2 μl RT-RamDA reaction solution, nuclease-free water] was treated at 95°C for 1 minute to activate the enzyme, and then 40 cycles of denaturation at 95°C for 15 seconds and extension at 60°C for 1 minute were performed.

Melting curve analysis was performed at 95°C for 15 seconds, 60°C for 15 seconds, and 95°C for 15 seconds.
The messenger RNA (mRNA) of β-actin was used as the target gene.

The primers for each gene are as follows:
βactin (mRNA)
Forward primer: CAGCTGAGAGGGGAAATCGTG (SEQ ID NO: 1)
Reverse primer: CGTTGCCAATAGTGATGACC (SEQ ID NO: 2)

The results are shown in Table 2 and FIG.

As shown in the results in Table 1 and Figure 1 above, under condition 1, in which guanidine thiocyanate was not added, an amplification curve was generated that could not be calculated as a Ct value, indicating nonspecific nucleic acid amplification in the RT-RamDA reaction and making quantitative measurement impossible. On the other hand, under conditions 2 to 4, in which guanidine thiocyanate was added, stable amplification curves were obtained. The PCR efficiency was also closer to 100% under conditions 2 to 4 compared to condition 1, suggesting that nonspecific amplification products were suppressed. This demonstrates that guanidine thiocyanate suppresses nonspecific amplification in the RT-RamDA reaction. Furthermore, these results confirm that when the amount of template RNA is relatively high (e.g., when the amount of template RNA is 100 pg to 10 ng), a lower amount of guanidine thiocyanate (e.g., 15 mM or less, or 10 mM or less) results in a lower Ct value and better results.

The above-disclosed embodiments and examples are all illustrative and not restrictive. Furthermore, embodiments and examples that combine the contents disclosed in the embodiments and examples are also included within the scope of the present invention. The technical scope of the present invention is defined by the claims, and includes all changes, modifications, substitutions, etc. that are equivalent in meaning to and within the scope of the claims.

The present invention effectively suppresses nonspecific nucleic acid amplification in the RT-RamDA method, which enables the detection of low-expression genes from minute amounts of RNA and an expansion of the number of detectable genes, thereby enabling more accurate and stable RNA analysis.

Claims (12)

A method for suppressing nonspecific nucleic acid amplification in an RT-RamDA reaction, characterized by allowing template RNA and a chaotropic agent to coexist in the RT-RamDA reaction solution. The method of claim 1, wherein a template RNA-containing biological sample to be subjected to an RT-RamDA reaction is treated with a cell lysis agent containing a chaotropic agent to prepare a template RNA-containing biological sample solution containing a chaotropic agent, and the template RNA-containing biological sample solution containing the chaotropic agent is mixed with an RT-RamDA reaction solution, thereby allowing the template RNA and the chaotropic agent to coexist in the RT-RamDA reaction solution. The method of claim 1 or 2, wherein the final concentration of the chaotropic agent in the RT-RamDA reaction solution is greater than 0 mM and less than or equal to 50 mM. The method of any one of claims 1 to 3, wherein the final concentration of the chaotropic agent in the RT-RamDA reaction solution is greater than 0 mM and less than or equal to 20 mM. The method of any one of claims 1 to 4, wherein the chaotropic agent is at least one chaotropic agent selected from the group consisting of guanidinium ions, urea ions, iodide ions, lithium ions, and salts thereof. The method of any one of claims 1 to 5, wherein the chaotropic agent is a guanidinium salt. The method of any one of claims 1 to 6, wherein the chaotropic agent is guanidine thiocyanate. The method of any one of claims 1 to 7, wherein the RT-RamDA reaction solution further contains an inorganic salt. The method of claim 8, wherein the inorganic salt is at least one selected from the group consisting of potassium salts, manganese salts, and magnesium salts. A method for stabilizing an RT-RamDA reaction, characterized by allowing template RNA and a chaotropic agent to coexist in the RT-RamDA reaction solution. A kit for use in the method of any one of claims 1 to 10, comprising an RT-RamDA reaction solution containing a chaotropic agent. A kit for use in the method of any one of claims 1 to 10, comprising a cell lysis agent containing a chaotropic agent.