KR20130069384A - Method for generating dna from rna in a sample and use thereof - Google Patents

Abstract

A method for efficiently generating DNA from RNA in a sample, a method for efficiently estimating the amount of RNA in a sample, and a composition for efficiently generating DNA from RNA in a sample are provided.

Description

Method for generating DNA from RNA in a sample and use according to the present invention.

A method for efficiently generating DNA from RNA in a sample, a method for efficiently estimating the amount of RNA in a sample, and a composition for efficiently generating DNA from RNA in a sample.

Amplifying small amounts of mRNA from a limited set of biological samples is the most fundamental and important task in gene analysis. Quantitative analysis of mRNA, such as gene-expression analysis, and qualitative analysis of mRNA, such as sequencing, are a major way to clarify the state of current biological tissues. The method of amplifying the amount of mRNA necessary enough and without change makes this analysis more accurate and meaningful.

The analysis of mRNA is a tool for research in biology, and it is a means for discovering drug targets for diagnosis of new drugs, discovering diagnostic markers, and observing mutations in individual genes, and is caused by genetic mutations such as genetic diseases and epigenetic diseases such as cancer. It can be used in various fields such as diagnosis of diseases.

Generating or amplifying DNA from RNA includes cDNA synthesis by reverse transcription and synthesizing DNA from cDNA. The reverse transcription process is accomplished by binding a common primer to an RNA template and catalyzing the nucleotide extension from the 3'-OH of the primer. As the primer used for reverse transcription, a primer having an oligo dT sequence or a random sequence is used. However, RNA has various one-dimensional sequences and two- and three-dimensional structures, so that the binding of primers cannot bind to all RNA sites with the same probability. Synthesis is also influenced by structural differences, which can lead to bias in which some RNAs are better synthesized in DNA, and in some cases, the synthesis is inhibited in the reverse transcription process where mRNA is converted into cDNA. In other words, when a plurality of RNAs are included, some of the RNAs may preferentially be amplified with DNA.

In addition, the process of synthesizing DNA from cDNA may use DNA dependent DNA polymerization reactions such as PCR and multiple displacement amplification (MDA). In this process, because of the diversity of the structure and sequence of the single-stranded cDNA, the efficiency of primer binding may vary from target to target, and thus there may be an amplification bias.

According to the prior art, DNA amplified from RNA has a problem in that it is biased generation or amplification.

One aspect provides a method for efficiently generating DNA from RNA in a sample.

Another aspect provides a method for efficiently estimating the amount of RNA in a sample.

Another aspect provides a composition for efficiently producing DNA from RNA in a sample.

One aspect comprises incubating a sample comprising one or more RNAs in the presence of RNA ligase to connect the 5 'and 3' ends of the RNA to form cyclized RNA; Microcompartment of a water-in-oil emulsion containing an aqueous component comprising cyclized RNA, a primer hybridizing to some region of the RNA or a sequence complementary to the RNA, and an RNA-dependent DNA polymerase and a DNA dependent DNA polymerase Introducing into; And generating DNA from the circularized RNA. It provides a method for amplifying DNA from RNA in a sample.

The method includes incubating a sample comprising one or more RNAs in the presence of RNA ligase to connect the 5 'and 3' ends of the RNA to form cyclized RNA.

The linkage may be a linkage within the same molecule. The RNA may be selected from the group consisting of mRNA, tRNA, rRNA and combinations thereof. The sample may include only RNA as a nucleic acid or an extract in which RNA is concentrated. It may be to include RNA separated by a RNA separation process from a biological sample. The RNA may be one that is synthetic, semisynthetic or transcript expressed from cells or viruses. The RNA may be one stored RNA. The storage may be stored by a known method. The storage may be stored for 1 year or more, for example, 1 year to 10 years. The RNA may be RNA derived from tissue stored in a frozen storage or formalin fixed paraffin embedded tissue at room temperature. It is known to isolate RNA from biological samples. For example, a trizol method or the like can be used.

The sample may include a degraded product of RNA isolated from a biological sample. The sample may be one that contains RNA isolated from a formalin-fixed paraffin-embedded (FFPE) tissue sample. The native mRNA of the eukaryotic cell may have a 5'-cap structure and a 3'-poly (adenylate) sequence. However, mRNA may be degraded during storage or treatment of the biological sample or mRNA isolated therefrom. In this case, the isolated product may not have the 5'-cap structure and 3'-poly (adenylate) sequence of the native mRNA structure. MRNAs that can be used for amplification in a method according to one aspect include those that do not have the structure of native mRNA as described above. The sample comprises RNA having a 5'-cap and 3'-OH; RNA with 5'-cap and 3'-monophosphate; RNA with 5'-OH and 3'-monophosphate; RNA with 5'-0H and 3'-OH; RNA with 5'-monophosphate and 3'-OH; And RNA having 5'-monophosphate and 3'-monophosphate.

The 5'-cap structure is a structure in which 7-methylguanylate is linked to 5'-OH of a 5'-terminal sugar by a triphosphate linkage or as a degradation product thereof, and a guanylate is 5'-OH of a 5'-terminal sugar. And a structure linked by a triphosphate linkage. The 3'-OH of the terminal guanylate of the 5'-cap structure, and / or 2'-OH and the 2'-OH of the first and second nucleotides from the terminal may be methylated.

The RNA may be modified to have 5'-phosphorylation and 3'-OH or to have 5'-phosphorylation and 3'-OH. The RNA may be one having a length of 10-100 nt, for example 19-40 nt.

The RNA ligase may be one capable of catalyzing the ligation. The RNA ligase may be, for example, T4 RNA ligase 1, T4 RNA ligase 2, Circligase, or a combination thereof.

The incubation may be carried out under conditions suitable for the activity of the RNA ligase. The incubation can be carried out in the presence of elements necessary for the activity of RNA ligase.

The method comprises a water-in-oil emulsion comprising an aqueous component comprising the one or more cyclized RNAs, a primer that hybridizes to some region of the RNA or a sequence complementary to the RNA, RNA-dependent DNA polymerase and DNA-dependent DNA polymerase. Introducing into the microcompartments of the.

It is known in the art to introduce aqueous components into microcompartments of water-in-oil emulsions. For example, by mixing the aqueous component with the oily component, the aqueous component can be included in the water-in-oil type microcompartment to be formed. Microcompartments may be used interchangeably with droplets. The microcompartment may have an average diameter of 10 μm or less. For example, it may be one having an average diameter of 100nm to 10μm, 100nm to 5μm, 100nm to 3μm, or 100nm to 2μm.

The said oily component shows the lipophilic component which is not mixed with water. The oily component may comprise a mineral oil, for example silicone oil.

The emulsion comprising the microcompartments may further comprise a surfactant in addition to the oily component. The surfactant can stabilize the microcompartment or emulsion state in the emulsion. The surfactant may increase the thermal stability of the emulsion. The surfactant is also called an emulsifier. The surfactant may comprise one or more selected from the group consisting of egg yolk, lecithin, sodium stearoyl lactylate, emulsifying wax, polysorbate 20, and cetereth 20. The surfactant may be a nonionic surfactant. The nonionic surfactant may be a hydrophilic lipophilic (HLB) having a nonionic surfactant of 4 or less. The HLB value can be calculated by the following Griffin equation.

HLB value = 20 × MH / M (MH: molecular weight of hydrophilic moiety, M: molecular weight of surfactant)

The nonionic surfactants include Span 80 (monooleate sorbitan: Fluka, Japan), Tween 80 (polyoxyethylene sorbitan monooleate, Nakarai, Japan), Triton X-100 (t-octylphenoxypolyethoxy Ethanol), sun soft No. At least one selected from the group consisting of 818SK (condensed ricinoleic acid polyglycerin ester: solar chemistry, Japan), and sun soft O-30V (glyceric oleic acid: solar chemistry, Japan).

The circularized RNA may be sufficiently diluted to include 3 molecules or less, for example, 2 molecules or 1 molecule or less in the microcompartment.

The primer may be a primer having a sequence specific primer or a random sequence. The primer may be 10 to 100nt in length, for example, 10 to 50nt, 10 to 40nt, 10 to 30nt, or 15 to 30nt. The random primer may be 5 to 10nt in length, for example, 5nt, 6nt, 7nt, 8nt, 9nt or 10nt in length. The primer may be single stranded DNA. The primer may include only reverse primer R complementary to the RNA. Or the primer may include one or more of reverse primer R complementary to the RNA, and forward primer F complementary to the sequence complementary to the RNA.

RNA-dependent DNA polymerases include enzymes having RNA-dependent DNA polymerizing activity. The RNA-dependent DNA polymerase is used interchangeably with reverse transcriptase. RNA-dependent DNA polymerases are HIV-1 reverse transcriptase derived from human immunodeficiency virus type 1, M-MLV reverse transcriptase derived from Moloney murine leukemia virus, avian myeloblastosis viruses), AMV reverse transcriptase, HIV reverse transcriptase, or a combination thereof.

The DNA-dependent DNA polymerase includes an enzyme having DNA-dependent DNA polymerization. The DNA-dependent DNA polymerase may be one having strand substitution activity. The DNA-dependent DNA polymerase may be selected from the group consisting of Bst DNA polymerase, exonuclease minus, pyrophage 3173 polymerase, Tth polymerase, and combinations thereof. For example, the DNA polymerase may be one of Bst DNA polymerase and exonuclease minus. Bst DNA polymerase, exonuclease minus is 67 kDa Bacillus stearotermophilus (5 '->3' polymerase activity and strand substitution activity and not 3 '->5' exonuclease activity) stearothermophilus) is a DNA polymerase protein (large fragment). It also has reverse transcription activity. Bst DNA polymerase, exonuclease minus can be used for nucleic acid amplification, whole genome amplification, multiple displacement amplification, etc., including isothermal amplification. M-MLV, AMV, HIV reverse electron enzymes have reverse transcriptase activity, ribonuclease and DNA-dependent DNA polymerase activity. Bst DNA polymerase and exonuclease minus are known to have stronger DNA-dependent DNA polymerization activity than RNA-dependent DNA polymerization activity, and reverse transcription activity of synthesizing DNA from RNA is performed under conditions in which only RNA is present. If single-stranded DNA is formed, DNA dependent DNA polymerization activity may act. Thus, the primer may comprise only primers complementary to the RNA template, or may include primers complementary to the resulting single stranded DNA.

The aqueous component may further include a reverse transcription reaction, or a reagent for a DNA polymerization reaction. Reagents for reverse transcription or reagents for DNA polymerization include reagents necessary for RNA dependent DNA polymerization, and / or DNA dependent DNA polymerization. The reagent may comprise a reaction buffer, ribonucleotide triphosphate or deoxyribonucleotide, coenzyme or cofactor necessary for the activity of the polymerase.

The RNA may be present in an average of 3 molecules or less, for example, 2 molecules or less, or 1 molecule or less per microcompartment.

In one embodiment, the step of introducing comprises one or more circularized RNAs, primers that hybridize to some regions of the RNA or to sequences complementary to the RNAs, RNA-dependent DNA polymerases and DNA-dependent DNA polymerases. Preparing an aqueous component; And mixing the aqueous component, the oil component, and the nonionic surfactant to prepare a water-in-oil emulsion.

The preparation can be accomplished by mixing the cyclized RNA, primers, RNA-dependent DNA polymerase and DNA-dependent DNA polymerase in an aqueous medium such as water, PBS, or buffer. The buffer may be a DNA polymerase reaction buffer or a PCR reaction buffer. The RNA-dependent DNA polymerase and the DNA-dependent DNA polymerase may be used in an amount such that an average of one or more molecules may be included in each microcompart generated.

The step of preparing a water-in-oil emulsion can be accomplished by mixing the aqueous component, oily component, and nonionic surfactant. The mixing may or may not be stirred. In addition, the mixing may be made by the application of ultrasonic waves. The said aqueous component and an oil component are as above-mentioned. The nonionic surfactant may be a hydrophilic lipophilic (HLB) having a nonionic surfactant of 4 or less. HLB can be calculated by the Griffin formula described above. The nonionic surfactants include Span 80 (monooleate sorbitan: Fluka, Japan), Tween 80 (polyoxyethylene sorbitan monooleate, Nakarai, Japan), Triton X-100 (t-octylphenoxypolyethoxy Ethanol), sun soft No. At least one selected from the group consisting of 818SK (condensed ricinoleic acid polyglycerin ester: solar chemistry, Japan), and sun soft O-30V (glyceric oleic acid: solar chemistry, Japan).

In a method according to one aspect, the production is performed under conditions in which at least one of catalyzing the RNA-dependent DNA polymerization reaction by RNA-dependent DNA polymerase and the DNA-dependent DNA polymerization reaction by DNA-dependent DNA polymerase occurs. It may be done. The production may be in an emulsion containing the microcompartments. The production may be one that is performed for a sufficient time so that DNA amplification in each microcompartment reaches the saturation step. The time may be performed for 1 hour or more, for example, 1 hour to 10 hours, 1 hour to 5 hours, 2 hours to 5 hours, 1 hour to 3 hours, or 1 hour 30 minutes to 2 hours 30 minutes. have. The term "generating" includes amplifying DNA. The resulting product may be a multimeric nucleic acid comprising portions in which the same sequence is repeated.

The production may be at any temperature condition if at least one of the RNA dependent DNA synthesis and the DNA dependent DNA synthesis is made. The production may be made at 40 ℃ to 50 ℃. The production may be made in an isothermal condition. The production may be made in an isothermal condition at 40 ℃ to 50 ℃. "Isothermal condition" means a condition in which thermal cycling is not performed, and does not necessarily mean that it must be carried out at a specific temperature in all processes. The production can also take place under thermocycling conditions.

The production may be by strand displacement amplification (SDA), for example rolling circle amplification. The strand substitution amplification, e.g., rolling circle amplification, represents a process of unidirectional nucleic acid amplification capable of rapidly synthesizing multiple copies of DNA or RNA circular molecules such as plasmids, bacteriophage genomes, viroidal circular RNA genomes. . Some eukaryotic viruses amplify their DNA by rolling circle mechanisms. Rolling circle amplification involves attaching random or specific primers to single stranded circular nucleic acids and extending the sequence by template dependent attachment of nucleotides to the 3′-OH terminus of the primers. When the extended sequence meets the double-stranded region including the portion to which the primer is bound, the strand replacement activity of the DNA polymerase can be extended while replacing the single-stranded DNA from the template. The primer may be a moiety comprising 3′-OH produced by cleavage, eg, nicking, of a double stranded nucleic acid.

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Another aspect includes incubating a sample comprising one or more RNAs in the presence of RNA ligase to connect the 5 'and 3' ends of the RNA to form cyclized RNA; An aqueous component comprising the circularized RNA, a partial region of the circularized RNA or a primer hybridizing to a sequence complementary to the circularized RNA, an RNA-dependent DNA polymerase and a DNA-dependent DNA polymerase Introducing into the microcompartments of the emulsion; Generating DNA from the circularized RNA; And estimating the amount of RNA species in the sample from the amount of generated DNA species.

Forming a circularized RNA; Introducing the aqueous component into the microcompartments of the water-in-oil emulsion; And generating DNA from the circularized RNA is as described above.

The method includes estimating the amount of RNA species in the sample from the amount of DNA species produced. Methods of measuring the amount of DNA species produced are known to those skilled in the art. For example, each DNA may be isolated and measured by electrophoresis, or the amount of target DNA may be measured by quantitative PCR. The resulting DNA species correlates in amount and proportion with the RNA species. That is, because a small number of molecules of RNA are introduced into one microcompartment and amplified, the amount and proportion of RNA species can be measured without bias. This bias can be further reduced by running the amplification reaction until saturation. That is, DNA can be sufficiently amplified from RNA or DNA contained in each microcompartment, and reacted until space and enzymatic action are limited such that no further amplification can occur in each microcompartment.

The method may further comprise estimating the ratio of the amount of each RNA species in the sample from the ratio of the amount of each DNA species amplified.

Another aspect is an aqueous solution comprising one or more cyclized RNAs, primers that hybridize to some region of the cyclicd RNA or to sequences complementary to the cyclicd RNA, RNA-dependent DNA polymerases and DNA-dependent DNA polymerases. Provided are compositions for producing DNA from RNA in a sample, including microcompartments of water-in-oil emulsions with components.

The microcompartments and the elements constituting them are as described above. The composition may be an emulsion comprising microcompartments.

According to the method for generating DNA from RNA in a sample according to one aspect, it is possible to generate DNA from RNA with reduced bias.

According to a method for estimating the amount of RNA in a sample according to another aspect, it is possible to efficiently estimate the amount of RNA in the sample.

According to a composition for generating DNA from RNA in a sample according to another aspect, DNA can be efficiently generated from RNA in a sample.

1 is a photograph showing the results of electro-interlocking the amplification result.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One:

(One) RNA  Ready

(1.1) linear Double strand DNA  Short story

RT-PCR was performed using universal human reference RNA (Stratagene, CAT: # 74000) as a template to prepare three kinds of double-stranded DNA fragments.

Specifically, 1 μg of UHR RNA, 1 μl of target specific primer (reverse) of 2 pmole, 1 μl of 10 mM dNTP mix, and 13 μl of total volume of H ₂ O were added to ₂₀₀ μl, followed by incubation at 65 ° C. for 5 minutes. Cool on ice for 1 minute. Then 4 μl of 5 × first strand buffer (Invitrogen, Superscript ^TM III buffer), 1 μl of 0.1 M DTT, 1 μl of 40 U / μl of RNaseOUT (Invitrogen), and 1 μl of 200 U / μl SuperScript ^TM IIII reverse transcriptase (Invitrogen) Incubate at 45 ° C. for 30 minutes. SuperScript ^™ IIII reverse transcriptase is an engineered version of M-MLV RT with reduced RNaseH activity and increased thermal stability. This enzyme can be used to synthesize first strand cDNA at temperatures of up to 55 ° C.

Next, a total of 20 μl reaction mixture was prepared including 10 μl of 2 × HS Prime Taq premix (GeNet BIO), 0.5 μM forward primer, 0.5 μM reverse primer, 2 μl reverse transcriptase reactant, and 6 μl water. The reaction mixture was incubated at 94 ° C. for 10 minutes, repeated 30 cycles of 30 seconds at 94 ° C., 30 seconds at 55 ° C. and 30 seconds at 72 ° C., followed by incubation at 72 ° C. for 5 minutes. HS Prime Taq premixes include HS Prime Taq DNA polymerase, reaction buffers, dNTP mixtures, and protein stabilizers. HS Prime Taq DNA polymerase is designed for hot-start PCR. This enzyme is inactive at room temperature and functional activity is restored during incubation at 94 ° C. for 10 minutes.

Linear double stranded DNA fragments obtained from incubation products were identified by electrophoresis. The result was 128 bp Actin, 128 bp GUSB, and 125 bp TFRC DNA fragment. The obtained DNA fragment contained a T7 promoter region at the 5 'end portion.

(1.2) Fragments RNA  making

MRNA was obtained by in vitro transcription reaction from the linear double-stranded DNA fragment obtained in (1.1). Specifically, linear double-stranded DNA fragment obtained at 100 ng of (1.1), 2 μl T7 RNA polymerase (Invitrogen), 1 × reaction buffer (40 mM Tris-HCl, 6 mM MgCl ₂ , 10 mM DTT, 2 mM 50 μl of the reaction mixture, including spermidine, pH 7.9 at 25 ° C., 7.5 mM NTP (ATP, CTP, GTP and UTP), and 25 μl of water, were incubated at 37 ° C. for 2 hours.

The resulting RNA was confirmed by electrophoresis. The results confirmed 106 bp Actin, 106 bp GUSB, and 103 bp TFRC RNA. The deoxyoligonucleotide sequences used for RT-PCR are shown in Table 1.

designation SEQ ID NO: T7_actin-100mer-F One T7_actin-100mer-R 2 T7_GUSB-100mer-F 3 T7_GUSB-100mer-R 4 T7_TFRC-100mer-F 5 T7_TFRC-100mer-R 6 Actin TaqMan probe 7 GUSB TaqMan probe 8 TFRC TaqMan probe 9

(1.3) RNA Self-connection

RNA products synthesized through in vitro transcription were synthesized using 5'-pyrophosphohydrolase (R'HH, New England Biolaboratories) and CircLigase ^™ II ssDNA Ligase (Epicentre). Induction gave cyclized RNA. CircLigase ^™ II ssDNA Ligase is a thermostable enzyme that catalyzes the intramolecular linkage (ie linearization) of ssDNA substrates with 5'-monophosphate and 3'-hydrosil groups. Linear ssDNA and ssRNA of about 15 bases or more are circularized by Circligase II ssDNA Ligase.

Specifically, 3 μg of the RNA product synthesized in (1.2) and 10 μl NEB2 buffer (10 ×), 10 μl RppH enzyme (5 U / μl) and an amount of 100 μl total volume were mixed and incubated at 37 ° C. for 1 hour.

RppH treated RNA was purified from the reaction product using mirVana ™ miRNA Isolation Kit (Ambion). 300ng of RppH treated RNA, 2μl Circligase II buffer (10x), 1μl 50 mM MnCl ₂ , 4μl 5M betaine, 1μl RNasin (40 unit / μl), and 1μl Circligase II (100U / μl) to a total volume of 20μl The reaction mixture was obtained by mixing with an amount of water to be added. The reaction mixture was incubated at 60 ° C. for 1 hour to yield linearized RNA.

(2) Annular RNA Amplify from

Different amounts of cyclic RNA (Actin) were added to Tris-HCl (pH 8.0) 52.5 mM, KCl 70 mM, (NH ₄ ) ₂ SO4 8.4 mM, MgCl ₂ 14 mM, dNTP 1.4 mM, Tween 20 0.12%, random 6-mer primer 26 μM And a reaction mixture of 4.6 U / ul of Bst DNA polymerase to prepare a reaction mixture with a total volume of 50 ul, which was incubated. The amount of RNA used at this time was 16pg, 80pg, 400pg, 2ng, and 10ng, respectively. Incubation was performed for 90 minutes isothermal amplification at 45 ℃. The resulting reaction product was electrophoresed to quantify the amplified DNA.

RNA usage 16 pg 80 pg 400 pg 2ng 10ng Amplification factor 103437 23375 4887 857 173 CV (%) 4.8 0.8 5.6 9.9 14 Product amount (μg) 1.6 1.8 1.9 1.7 1.7 Concentration (ng / μl) 33.1 37.4 39.1 34.3 34.7

(3) Water type  Generation of drops

To 50 ml of mineral oil, add Span 80, tween 80, and Triton X-100 to 4.5% (v / v), 0.4% (v / v) and 0.05% (v / v), respectively. A surfactant mixture was obtained. Next, 400ul of oil-surfactant mixture was transferred to a cryotube vial and mixed with stirring at 1000rpm for 5 minutes using a 3x8 mm stir bar.

Aqueous phase comprising RNA synthesized in (1.3) (20 mM Tris-HCl (pH 8.8, 25 ° C.), 10 mM (NH ₄ ) ₂ SO ₄ , 10 mM KCl, 2 mM MgSO ₄ , 0.1% Triton X- 100 ul, 26 uM random hexamer, 1.3 mM dNTP, and 200 ul of 4.8 unit / ul Bst DNA polymerase were added drop wise to the freezing tube vial containing the oil-surfactant mixture and mixed with stirring at 1000 rpm for 5 minutes.

As a result, an emulsion containing the drops containing the aqueous phase was obtained. The resulting droplets had an average diameter of 3.33 μm (CV 40%).

(4) polymerization reaction

The emulsion was incubated at 45 ° C. for 5 hours to allow reverse transcription and DNA dependent DNA polymerization. After the reaction, RNase was added and incubated to degrade the remaining RNA.

Specifically, 2 ng of cyclic RNA (Actin) was 52.5 mM Tris-HCl (pH 8.0), KCl 70 mM, (NH ₄ ) ₂ SO ₄ 8.4 mM, MgCl ₂ 14 mM, dNTP 1.4 mM, Tween 20 0.12%, forward primer 1 μM Mixed with a solution of 1 μM reverse primer (or 26 μM random hexamer), and 4.6 U / ul of Bst DNA polymerase to prepare an aqueous volume of 200 ul of total volume, thereby producing water-in-oil droplets and This was incubated at 45 ° C. for 5 hours.

After completion of the reaction, the emulsion was centrifuged at 13,000 g for 5 minutes to remove the oil phase. 1 ml of saturated diethyl ether was added to the aqueous phase to break up the droplets of the emulsion and remove the mineral oil. The amplification was confirmed by performing electrophoresis on the product obtained in FIG.

1 is a photograph showing the results of electro-interlocking the amplification result. The reaction product was branched and amplified and observed in various sizes on electrophoresis images. Ie, amplified with multi-cancartimeric nucleic acids. In Figure 1, lanes 1 and 2 represent Actin gene specific primers, lanes 3 and 4 were amplified using a random hexamer, lanes 1 and 3 when RNA was not present in the sample, lanes 2, 4 is the case where cyclic RNA (Actin) is in a sample.

Example  2:

Three cyclic RNAs were mixed at a constant ratio of Actin: 5 pg, TFRC: 50 fg, and GUSB: 5 fg, and then the mixture was used as a sample, using Tris-HCl (pH 8.0) 52.5 mM, KCl 70 mM, (NH _4). ) ₂ SO ₄ 8.4 mM, MgCl ₂ 14 mM, dNTP 1.4 mM, Tween 20 0.12%, 26 μM random hexamer, and a solution of 4.6 U / ul of Bst DNA polymerase to prepare a reaction volume of 200 ul total volume, This resulted in the production of water-in-oil droplets, which were isothermally incubated at 45 ° C. for 15 hours (hereinafter referred to as “emulsion amplification”).

The obtained product was subjected to quantitative RT-PCR (qRT-PCR) in a PCR reaction solution containing primers specific for three types of RNA, respectively. Specifically, 0.1 μl of 100 μM forward primer, 0.1 μl of 100 μM reverse primer, 0.2 μl of 100 μM TaqMan ^™ probe, 10 μl of LIGHTCYCLER 480 PROBES MASTER (Roche), and 5 μl of cyclized product were added to a 100 μl tube. Was added, and qRT-PCR was performed on a Lightcycler 480 (Roche) instrument. The thermocycle was incubated with 50 cycles of 10 seconds at 95 ° C. and 10 seconds at 55 ° C.

For initial RNA quantification, add 3 μl of target specific primer (reverse), 1 μl of 10 mM dNTP mix, and 13 μl total volume to 3 RNA mixtures, then incubate at 65 ° C. for 5 minutes and then on ice 1 Cool for minutes. Final volume was then added with 4 μl of 5x first strand buffer (Invitrogen, Superscript ^TM III buffer), 1 μl of 0.1 M DTT, 1 μl of RNaseOUT (Invitrogen) at 40 U / μl, and 1 μl of 200 U / μl SuperScriptTM III reverse transcriptase (Invitrogen) After addition to 20 μl, incubation at 45 ° C. for 30 minutes yielded the cDNA product. 5 μl of the resulting cDNA product was analyzed in the same manner as qPCR of the amplification product.

When amplifying each RNA single molecule in a single emulsion, the competition reaction is minimized so that the Pearson correlation of Ct values before and after amplification is 0.99, which is almost the same.

Table 3 shows the DNA amplification products obtained as a result of qRT-PCR on samples before emulsion amplification and after samples.

Before amplification (Ct) After amplification (Ct) Actin 25.99 23.60 TFRC 30.78 28.74 GUSB 33.06 30.21 P.Correlation 0.99

As shown in Table 3, the three kinds of DNA amplification products obtained were present in almost the same ratio as the RNA initially added.

<110> Samsung Electronics Co., Ltd. <120> Method for generating DNA from RNA in a sample and use <130> PN098693 <150> KR 102011013775 <151> 2011-12-15 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> T7_actin-100mer-F <400> 1 gaaattaata cgactcacta tacctggcct cgctgtccac 40 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> T7_actin-100mer-R <400> 2 gtcatagtcc gcctagaagc 20 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> T7_GUSB-100mer-F <400> 3 gaaattaata cgactcacta taccaggtat ccccactcag 40 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> T7_GUSB-100mer-R <400> 4 cgccctgact cggggagg 18 <210> 5 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> T7_TFRC-100mer-F <400> 5 gaaattaata cgactcacta tacctggact atgagaggta c 41 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> T7_TFRC-100mer-R <400> 6 cagccactgt aaactcaggc c 21 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Actin TaqMan probe: FAM is attached to 5 'end and BHQ1 is          attached to 3 'end. <400> 7 aggagtatga cgagtccggc ccc 23 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GUSB TaqMan probe: FAM is attached to 5 'end and BHQ1 is          attached to 3 'end. <400> 8 tcaagtaaac gggctgtttt ccaaaca 27 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> TFRC TaqMan probe: FAM is attached to 5 'end and BHQ1 is attached          to 3 'end. <400> 9 agggatctga accaatacag agcagaca 28

Claims (17)

Incubating a sample comprising one or more RNAs in the presence of RNA ligase to connect the 5 'and 3' ends of the RNA to form cyclic RNA;
Aqueous components comprising cyclized RNA, primers that hybridize to some region of the cyclized RNA or to sequences complementary to the cyclized RNA, RNA-dependent DNA polymerase and DNA dependent DNA polymerase may be used in the water-in-oil emulsion. Introducing into a microcompartment; And
Generating DNA from the circularized RNA; comprising; amplifying DNA from RNA in a sample. The method of claim 1, wherein the RNA is mRNA, tRNA, rRNA, miRNA or a combination thereof. The method of claim 1, wherein the RNA is a transcript expressed from a cell or a virus. The method of claim 1, wherein the RNA ligase is Circligase. The method of claim 1, wherein the RNA-dependent DNA polymerase, DNA dependent DNA polymerase or combinations thereof has strand substitution activity. The method of claim 1, wherein the amplification is strand substituted amplification. The method of claim 1, wherein the aqueous component further comprises a reverse transcription reaction reagent or a reagent required for DNA polymerization. The method of claim 1, wherein the RNA is present on average less than one molecule per microcompartment. The method of claim 1, wherein the introducing step comprises circularized RNA, primers that hybridize to a region of the RNA or to a sequence complementary to the RNA, and an RNA-dependent DNA polymerase and a DNA-dependent DNA polymerase. Preparing an aqueous component; And
Mixing the aqueous component, the oil component, and the nonionic surfactant to prepare a water-in-oil emulsion. The method of claim 9, wherein the nonionic surfactant is a nonionic surfactant having a hydrophilic lipophilic balance (HLB) of 4 or less. The method of claim 1, wherein the amplification is performed under conditions that catalyze by one or more of RNA-dependent DNA polymerase and DNA dependent DNA polymerase. The method of claim 1, wherein the amplification is performed for a time sufficient to allow DNA amplification in each microcompartment to reach saturation. The method of claim 1, wherein said amplification is performed at isothermal. The method of claim 1 wherein the amplification is carried out at a temperature of 40 ℃ to 50 ℃. Incubating a sample comprising one or more RNAs in the presence of RNA ligase to connect the 5 'and 3' ends of the RNA to form cyclic RNA;
Aqueous components comprising cyclized RNA, primers that hybridize to some region of the cyclized RNA or to sequences complementary to the cyclized RNA, RNA-dependent DNA polymerase and DNA dependent DNA polymerase may be used in the water-in-oil emulsion. Introducing into the microcompartments;
Generating DNA from the circularized RNA; And
Estimating the amount of RNA in the sample from the amount of DNA species produced. The method of claim 15, further comprising estimating the ratio of the amount of each RNA species in the sample from the ratio of the amount of each DNA species amplified. Micro of water-in-oil emulsions comprising an aqueous component comprising at least one circularized RNA, a primer hybridizing to a partial region of the RNA or to a region complementary to the RNA, RNA-dependent DNA polymerase and DNA-dependent DNA polymerase A composition for producing DNA from RNA in a sample, comprising a compartment.

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