KR102034721B1

KR102034721B1 - Method for determining a ratio of RNA type in a sample

Info

Publication number: KR102034721B1
Application number: KR1020120130504A
Authority: KR
Inventors: 김세희; 이주원; 최고봉
Original assignee: 삼성전자주식회사
Priority date: 2011-12-15
Filing date: 2012-11-16
Publication date: 2019-10-22
Also published as: KR20130069383A

Abstract

Provided is a method of generating DNA from RNA in a sample using circular RNA.

Description

Method for determining a ratio of RNA type in a sample

A method for amplifying target RNA in a sample with DNA without bias to determine the proportion of RNA species present in the sample.

Transcriptome analysis is an important tool for diagnosing disease and finding the cause, and is a basic research method for discovering targets for drug development. All transcriptome analysis requires the amplification of small amounts of RNA from samples.

RNA expressed from a cell or virus comprises a native active form, for example, a 5'-cap structure and a 3'-poly (adenylate) sequence for mRNA of eukaryotic cells. However, these sequences may be removed or modified during the storage or analysis of biological samples. For example, RNA isolated from formalin-fixed parafin-embedded (FFPE) tissue that has been frozen or stored at room temperature is present in a size of about 300 bp or less. Storage of FFPE tissue is a method of storing a tissue sample of a patient, and is commonly used because it is inexpensive and simple to store at room temperature. Because FFPE samples have accumulated in many patients over time, retrospective studies of these patient samples are possible. As described above, although gene expression profile studies using FFPE samples are important tools in various disease studies including cancer studies, RNAs isolated from FFPE tissue samples mostly exist as fragmented RNAs.

Conventional methods of analyzing RNA include reverse transcription of RNA to generate cDNA, and amplification products obtained by amplifying the cDNA. In this method, in the process of reverse transcription of RNA to generate cDNA, specific target RNA may be preferentially converted to cDNA, and in the case of poly (A) dependent cDNA synthesis, fragmented RNA may not be converted to cDNA and may be lost do. Therefore, there is a need for a method for generating a DNA which does not lose the ratio of the RNA species in the sample and does not lose it. There is also a need for a method for efficiently determining the abundance ratio of RNA species in a sample.

Provided are methods for efficiently determining the proportion of RNA species present in a sample by amplifying DNA directly from RNA in the sample without biasing the proportion of target RNA species in the sample.

One aspect includes providing a sample comprising a plurality of types of RNA; Incubating the sample in the presence of an enzyme that converts the 5'-cap structure to 5'-monophosphate to convert RNA having the 5'-cap structure to RNA having 5'-monophosphate; Incubating the sample in the presence of an enzyme that phosphorylates 5'-OH, converting RNA with 5'-OH to RNA with 5'-monophosphate; Incubating the sample in the presence of an enzyme that dephosphorylates the phosphorylated 3'-terminus to convert RNA having a 3'-phosphate group to RNA having a 3'-OH; Incubating the sample in the presence of RNA ligase to produce circular RNA; And incubating the sample in the presence of RNA-dependent DNA polymerase to generate DNA from circular RNA; Identifying the generated DNA; And determining a ratio of RNA species in the sample from the identified DNA.

The method includes providing a sample comprising a plurality of types of RNA. The RNA may be natural, synthetic or semisynthetic mRNA, tRNA, or rRNA. The sample may comprise a biological sample or RNA isolated therefrom. The biological sample includes a virus or a biological sample. For example, the sample may be selected from the group consisting of blood, saliva, urine, feces, tissues, cells and biopsies. A plurality of kinds of RNA means that two or more RNAs of different lengths, sequence compositions or structures are included. For example, two or more RNA molecules having different lengths or sequence compositions, even if they have the same mRNA structure. The sample may be, for example, a whole transcript or part thereof. It is known to isolate RNA from biological samples. For example, the Trizol method can be used.

In addition, the sample may include a stored biological sample or RNA isolated therefrom. The storage may be stored by a known method. For example, cryopreservation or formalin-fixed paraffin-embedded (FFPE) tissue may be stored at room temperature.

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 eukaryotic cells has a 5'-cap structure and a 3'-poly (adenylate) sequence. However, mRNA can be degraded during storage or processing of biological samples 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 (cap) is a structure in which 7-methylguanylate is linked to the 5'-OH of the 5'-terminal sugar by a triphosphate linkage or as a degradation product thereof, wherein the guanylate is a 5'-terminal sugar. '-OH includes 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 method includes incubating the sample in the presence of an enzyme that converts the 5'-cap structure to 5'-monophosphate, thereby converting the RNA having the 5'-cap structure to the RNA having the 5'-monophosphate. .

The enzyme for converting the 5'-cap structure to 5'-monophosphate may be one or more selected from the group consisting of tobacco acid pyrophosphatase (TAP), Rai1, Dom3Z, yeast Dcp1 and Dcp2, human Dcp1 and Dcp2. The incubation can be done under conditions suitable for converting the 5'-cap structure to 5'-monophosphate. Such conditions can be appropriately selected by those skilled in the art depending on the enzyme selected. TAP is an enzyme with one activity that catalyzes the hydrolysis of phosphoric ester bonds at the 5'-end of mRNA. The incubation can be performed using TAP 10 × reaction buffer: 0.5M sodium acetate, pH 6.0, 10 mM EDTA, 1% β-mercaptoethanol, and 0.1% Triton X-100.

The method includes incubating the sample in the presence of an enzyme that phosphorylates 5'-OH, thereby converting RNA with 5'-OH to RNA with 5'-monophosphate. Enzymes that phosphorylate 5'-OH may be enzymes known to phosphorylate 5'-OH, for example kinases. For example, the enzyme that phosphorylates 5'-OH can be a polynucleotide kinase (PNK), for example T4 polynucleotide kinase (T4 PNK) or a variant thereof. Such conditions can be appropriately selected by those skilled in the art depending on the enzyme selected. The incubation can be done under conditions suitable for phosphorylating 5'-OH. PNK catalyzes the transfer and exchange of polynucleotides (double-stranded and single-stranded DNA and RNA) of phosphoric acid at the γ position of ATP and the 5'-hydroxyl end of the nucleoside 3'-monophosphate. PNK can also catalyze the removal of 3'-phosphoryl groups from 3'-phosphoryl polynucleotides, deoxynucleoside 3'-monophosphates and deoxynucleoside 3'-diphosphates. Therefore, when using PNK, phosphorylation of 5'-OH and removal of 3'-phosphoryl group can be performed simultaneously or in the same reaction process.

The method includes incubating the sample in the presence of an enzyme that dephosphorylates the phosphorylated 3'-terminus, converting RNA with 3'-phosphate groups to RNA with 3'-OH. The enzyme that dephosphorylates the phosphorylated 3'-terminus may be an enzyme known to dephosphorylate the 3'-terminal phosphate group, for example phosphatase. The enzyme that dephosphorylates the phosphorylated 3′-terminus may be T4 polynucleotide kinase (PNK). The incubation can be made under conditions suitable for dephosphorylation of the phosphorylated 3'-terminus. Such conditions can be appropriately selected by those skilled in the art depending on the enzyme selected. The phosphorylated 3′-end may be triphosphorylated, diphosphorylated, or monophosphorylated.

The method includes incubating the sample in the presence of RNA ligase to produce circular RNA. RNA ligase can be an enzyme known in the art. RNA ligase may be, for example, a catalyst for intramolecular linking, ie self-ligation. RNA ligase can be, for example, T4 RNA ligase 1, T4 RNA ligase 2, CircLigase I, CircLigaseII, Mth RNA ligase, or a combination thereof. The incubation can be made under conditions suitable for connecting the 5'-monophosphate ends and the 3'-OH ends to each other. Such conditions can be appropriately selected by those skilled in the art depending on the enzyme selected. The connection may be a self connection. T4 RNA ligase can template-independently catalyze the formation of phosphodiester bonds between the 5'-phosphoryl-terminated nucleic acid donor and the 3'-hydroxyl-terminated nucleic acid receptor. T4 RNA ligase is ATP dependent and active against a wide range of substrates including RNA, DNA, oligoribonucleotides, oligodeoxyribonucleotides, and many nucleotide inducing agents. Incubation can be performed using T4 RNA ligase 10 × reaction buffer: 330 mM Tris-acetate (pH 7.5), 660 mM potassium acetate, 100 mM magnesium acetate, and 5 mM DTT.

The method includes incubating the sample in the presence of RNA-dependent DNA polymerase to generate DNA from circular RNA. The RNA-dependent DNA polymerase may be one having only reverse transcriptase activity or one having DNA polymerase activity together. The RNA-dependent DNA polymerase may be one having strand substituted DNA polymerase activity. That is, it may be one having not only reverse transcription activity but also DNA-dependent DNA polymerase activity. RNA-dependent DNA polymerase may be Bst DNA polymerase, exonuclease minus, Tth DNA polymerase, PyroPhage ^™ 3173 DNA polymerase, BcaBEST DNA polymerase, or a combination thereof. For example, the RNA-dependent DNA polymerase may be one that is Bst DNA polymerase, exonuclease minus. Bst DNA polymerase, exonuclease minus 67 kDa Bacillus stearothermophilus with 5'-3 'polymerase activity and strand substitution activity and no 3'-5' exonuclease activity 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 (MDA), and the like, including isothermal amplification. The incubation can be made under conditions suitable for reverse transcription and / or DNA-dependent DNA polymerization. Such conditions can be appropriately selected by those skilled in the art depending on the enzyme selected. Generating DNA includes amplifying the DNA. The amplification may be performed by rolling circle amplification. Amplification Rolling circle amplification refers to the 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. The amplification may be performed in the presence of random primers or sequence specific primers. The random primer may be one having a random nucleotide sequence of 5-15nt in length, for example 6nt. Generating DNA from circular RNA may be incubating in the presence of a separate DNA-dependent DNA polymerase, in addition to an RNA-dependent DNA polymerase.

In one embodiment, the method may further comprise incubating the sample in the presence of poly (adenylate) polymerase prior to the circular RNA production step to add a poly adenylate sequence at the 3′-end. By adding the poly adenylate sequence to the 3'-end, the formed circular RNA can have a poly adenylate sequence, regardless of its sequence composition. Therefore, by using a sequence complementary to the poly adenylate sequence, for example, an oligo dT sequence, as a primer in the amplification step, all the RNA present in the sample can be amplified. Since this amplification is formed to correspond to the proportion of the original RNA present in the original sample, the amplification product can also be obtained corresponding to the proportion of the RNA species present in the original sample. Thus, amplification may be performed in the presence of oligo dT primers. The oligo dT primer may have a length of 10 to 100nt, for example, 10 to 50nt. The poly (adenylate) polymerase may be E. coli poly (A) polymerase I.

According to the method for generating DNA from RNA in a sample according to one embodiment, DNA may be generated from target RNA so as to have high similarity with a ratio of a plurality of types of RNA in the sample. That is, the target RNA can be converted to DNA evenly without preferentially converting only specific target RNA to DNA. This is believed to be due to being able to convert all possible target RNAs into circular RNAs, but is not limited to specific mechanisms.

In the method, the steps may be performed sequentially or all or part of the steps may be performed simultaneously.

The method may further comprise introducing the circular RNA, and the elements necessary for nucleic acid polymerization, into the droplets after generating the circular RNA. Elements required for nucleic acid polymerization may include reverse transcriptase, DNA-dependent DNA polymerase, cofactors, buffers, or a combination thereof. Generating DNA from circular RNA may be performed at isothermal. Generating DNA from the circular RNA may be performed without thermocycling at 40 to 50 ℃.

The method comprises an aqueous component comprising one or more of the at least one circularized RNA, a primer that hybridizes to a region of the RNA or to a sequence complementary to the RNA, an RNA-dependent DNA polymerase and a DNA-dependent DNA polymerase. Introducing into the microcompartments of the water-in-oil emulsion. The microcompartments may be droplets.

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).

The method includes identifying the resulting DNA. The confirmation may include identifying the amount of DNA as well as confirming the DNA sequence. Identifying the amount of DNA can be done by known methods. For example, it can be done by spectroscopic methods, electrical methods, methods of labeling using a detectable label and detecting DNA, or a combination thereof. Identification of the sequence of DNA can be accomplished by known methods. Identification of the sequence of the DNA can be made by, for example, chemical sequencing methods, electrical sequencing methods, hybridization prevention or a combination thereof. Chemical sequencing methods may be the Maxam-Gilbert method, chain-termination methods, or a combination thereof.

The method also includes determining a ratio of RNA species in the sample from the identified DNA. The determination may be based on the amount of each DNA type identified, presuming that each corresponding RNA type is present.

According to the above method, the RNA type in the sample can be converted into DNA without deflection, and the ratio of RNA present in the sample can be efficiently determined.

1 is a photograph showing the results of electrophoresis of the PCR product.
Figure 2 is a photograph showing the result of electrophoresis of the product in vitro.
Figure 3 is a photograph showing the results of electrophoresis of the self-connected reaction product.
Figure 4 is a photograph showing the results of electrophoresis of the product amplified by MDA from circular RNA.
Figure 5 is a photograph showing the results of electrophoresis of the PCR product.
6 is a photograph showing the results of electrophoresis of the transcription product in vitro.
Figure 7 is a photograph showing the results of electrophoresis of the self-connected reaction product.
8 shows the results of electrophoresis of the emulsion amplification products.
9 is a photograph showing the results of electrophoresis of the product obtained by incubating the trimming reaction product and the self-linked product in the presence of XRN1 and RNaseR, respectively.

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 1: generated by in-vitro transcription RNA of Annular ( circularization ) And amplification

(One) RNA Preparation

DNA containing the T7 promoter sequence was amplified by PCR using pRL-CMV vector plasmid DNA as a template and a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2 as primers. Using the Megascript ^TM T7 Kit (Ambion) was prepared using mRNA from the PCR product containing the T7 promoter sequence. Specifically, 100 ng of amplification products, 2 μL ATP, CTP, UTP, GTP solution, and 2 μL of enzyme mixture (mix), respectively, were mixed at a final volume of 20 μL under 1 × reaction buffer conditions and incubated at 37 ° C. for 2 hours. As a result, 100 nt RNA was obtained by in vitro transcription. 1 is a photograph showing the results of electrophoresis of the PCR product. Figure 2 is a photograph showing the result of electrophoresis of the product in vitro.

(2) terminal Trim

MRNA synthesized in the in vitro transcription process of (1) has a triphosphate at the 5 'end. Synthesized 300 ng of RNA in RNA 5 'RppH in 5' Pyrophosphatase (RppH) reaction buffer (50 mM NaCl, 10 mM Tris-Cl, 10 mM MgCl ₂ , 1 mM dithiothreitol (DTT), pH7.9) Incubation with 5 units at 37 ° C. for 30 minutes yielded RNA with 5′-monophosphate.

(3) self-connection

RngH-treated 300ng of RNA with 5'-monophosphate was added to MnCl ₂ and betaine in CircLigase II buffer (33mM Tris-acetate pH7.5, 66mM potassium acetate, 0.5mM DTT) with final concentrations of 2.5mM and 1M, respectively. After the addition, 100unit CircLigase II was added and incubated at 60 ° C for 1 hour. As a result, the 5'-monophosphate terminal and the 3'-OH terminal of the mRNA were linked to form a circular RNA. The obtained samples were loaded onto 10% TBE urea gel (Invitrogen) and electrophoresed to identify shifted circular RNA after self-linking.

Figure 3 is a photograph showing the results of electrophoresis of the self-connected reaction product. In FIG. 3, A is a sample before connection, and B is a sample after connection.

(4) RNA from DNA Amplification

DNA from the RNA using the primers (SEQ ID NO: 4) capable of binding the formed RNA to the DNA synthesized with enzyme and RNA template specific primer (SEQ ID NO: 3) having reverse transcription activity and strand-substituted DNA polymerase activity. Was synthesized.

Specifically, Bst DNA polymerase buffer (Tris-HCl pH8.0 52.5 mM, KCl 70 mM, (NH ₂ ) ₄ SO ₄ 8.4 mM, MgCl ₂ RNA was incubated for 2 hours at 45 ° C by adding 1.6μM of RNA template specific primers (SEQ ID NO: 3 and SEQ ID NO: 4) and 0.16 unit / μL of Bst DNA polymerase in the presence of 14 mM, dNTP 1.4 mM, Tween 20 0.12%). DNA was amplified from. As a result, DNA was amplified by multi-strand substitution amplification (MDA).

Figure 4 is a photograph showing the results of electrophoresis of the product amplified by MDA from circular RNA. In Figure 4,-, and + represent a control and a sample containing the 100 nt circular RNA, respectively. As shown in FIG. 4, DNA amplification products were present in the experimental group (+).

Example 2: 3 kinds obtained by in vitro transcription RNA of With circularization Amplification Ratio Measurement

(1) 3 types of standard ( reference ) RNA For compounding cDNA synthesis

1 μg UHRR (Universal Human Reference RNA, Stratagene: Cat Nr: 740000) was added to 1 × reverse transcriptase (RT) buffer (10 mM Tris-Cl pH8.3 at 25 ° C., 90 mM KCl), 1 mM MnCl ₂ , 0.2 mM dNTP mix, 15 pmol Each downstream primer (Actin: SEQ ID NO: 5, GUSB: SEQ ID NO: 6, and TFRC: SEQ ID NO: 7) to a final volume of 20 μL in the presence of 1 μL, 5 unit Tth DNA polymerase (Promega) and at 70 ° C. The first strand cDNA was synthesized by incubation for 20 minutes at.

20 μl of the first strand cDNA sample synthesized, 1 pL upstream primer (Actin: SEQ ID NO: 8, GUSB: SEQ ID NO: 9, and TFRC: SEQ ID NO: 10) containing a T7 promoter sequence for each 1 μL, 1x chelate buffer (10 mM Tris-Cl pH8.3, 100 mM KCl, 0.75 mM EGTA, 0.05% Tween 20, 5% Glycerol), and 0.25 mM MgCl ₂ were added and incubated at 95 ° C. for 5 minutes, followed by incubation at 70 ° C. for 20 minutes. Two strand cDNA was synthesized.

Next, PCR amplification was performed using the cDNA product as a template and the upstream and downstream primers as primers.

Next, 100 ng of the PCR amplification product was synthesized using a Megascript ^™ T7 Kit (Ambion) that induces transcription using T7 RNA polymerase. Specifically, a final volume of 100 ng of amplification product and 2 μL ATP, CTP, UTP, and GTP solutions, and 2 μL of enzyme mix, respectively, was mixed and incubated at 37 ° C. for 2 hours. Figure 5 is a photograph showing the results of electrophoresis of the PCR product. 6 is a photograph showing the results of electrophoresis of the transcription product in vitro. As shown in Fig. 6, three kinds of about 100 nt RNA were obtained by in vitro transcription.

(2) terminal Trim

The mRNA synthesized in the in vitro transcription process of (1) has a 5-terminal triphosphate. 300 ng of the synthesized RNA was incubated for 30 minutes at 37 ° C. with 5 'RNA 5' RppH in RppH reaction buffer (50 mM NaCl, 10 mM Tris-Cl, 10 mM MgCl ₂ , 1 mM dithiothreitol, pH7.9) RNA with monophosphate was obtained.

(3) self-linking reaction and annular RNA Separation

300 ng of RNA with 5'-monophosphate obtained by RppH treatment was treated in CircLigase II buffer (33 mM Tris-acetate pH7.5, 66 mM potassium acetate, 0.5 mM DTT) with final concentrations of 2.5 mM and 1 M, respectively, of MnCl ₂ and betaine. After the addition, 100unit CircLigase II was added and incubated at 60 ° C for 1 hour. As a result, the 5'-monophosphate terminal and the 3'-OH terminal of the mRNA were linked to form a circular RNA.

Only circular RNA was isolated to determine the correct amplification fold. 10% TBE urea gel (Invitrogen) was loaded with a sample subjected to self-linking reaction, followed by electrophoresis to cut a band of self-linked cyclic RNA.

Cyclic RNA was extracted from the gel using Midi GeBAflex-tube Gel Extraction & Dialysis Kit (Komabiotech). As a result, only three kinds of circular RNA were isolated.

Figure 7 is a photograph showing the results of electrophoresis of the self-connected reaction product. In FIG. 7, A shows a sample after connection and B shows a sample before connection.

(4) Emulsion Used Multi-strand Substitution amplification ( MDA )

(4.1) Water-in-oil type Drop Formation and Polymerization

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. An activator mixture was obtained. Next, 400 μl of the oil-surfactant mixture was transferred to a cryotube vial and mixed with stirring at 1000 rpm for 5 minutes using a 3 × 8 mm stir bar.

Three different aqueous phases were made to make each emulsion of three circular RNAs (Actin, GUSB, and TFRC gene RNA). Aqueous phase containing 2 ng of circular RNA (40 mM Tris-HCl pH 8.8, 6.4 mM (NH ₄ ) ₂ SO ₄ , 53 mM KCl, 10.6 mM MgSO ₄ , 0.1% Triton X-100, 30 μΜ 200 μl of random hexamer, 1.33 mM dNTPs, 4.8 unit / μl Bst DNA polymerase, 16 unit / μl M-MLV reverse-trancriptase, 40 ng / μL T4 SSB and 0.2% Antifoam) contains the oil-surfactant mixture Was added drop wise to a frozen tube vial and mixed with stirring at 1000 rpm for 5 minutes.

As a result, three different emulsions containing the drops containing the aqueous phase were obtained. The resulting droplets had an average diameter of 3.33 μm (CV 40%). The droplet together with the circular RNA, reverse transcription polymerase, DNA polymerase together with the components necessary for the polymerization reaction, the amplification reaction can be carried out in the droplet. Prototype RNA is sufficiently diluted to contain less than 50% of the total drop.

(4.2) Polymerization

The emulsion was incubated at 45 ° C. for 3, 6, 9, 12, and 15 hours, respectively, to allow reverse transcription and DNA dependent DNA polymerization. After the reaction, RNase was added and incubated to degrade the remaining RNA.

After completion of the reaction, the emulsion was centrifuged at 13,000 g for 5 minutes to remove the oil phase. 1 ml of diethyl ether was added to the aqueous phase to break up the droplets of the emulsion and remove the mineral oil. The amplified DNA was confirmed by electrophoresis, and the amplification product was identified using Qubit ^™ dsDNA HS Assay Kit (Invitrogen).

8 shows the results of electrophoresis of the emulsion amplification products. As shown in FIG. 8, it was confirmed that amplification products existed at reactions 3, 6, 9, 12, and 15 hours.

The DNA in the emulsion amplification product was quantified using the Qubit ^™ dsDNA HS Assay Kit (Invitrogen).

Amplification time (hours) Actin (μg) GUSB (μg) TFRC (μg) 3 40.3 26.0 18.5 6 60.9 48.4 40.6 9 62.7 54.4 44.3 12 62.6 54.8 49.4 15 63.2 52.7 54.2

As shown in Table 1, it was confirmed that the initial 2ng RNA was amplified about 10 ⁴ times. As a result, the linearized RNA could be amplified with high amplification efficiency by MDA reaction.

Example 3. Fragmented UHRR Whole with Transcript Amplification

(One) RNA Preparation: Fragmented FFPE RNA copy

RNA was fragmented by adding 4 μg of universal human reference RNA (UHRR, stratagene) to 50 μl of H ₂ O and irradiating for 5 minutes with a Covaris Sonicator.

(2) terminal Trim

2 μg of fragmented RNA was added to 25 unit TAP in Tobacco Acid Pyrophosphatase (TAP) reaction buffer (50 mM sodium acetate pH6.0, 1 mM EDTA, 0.1% β-mercaptoethanol, 0.01% Triton X-100). Addition and incubation at 37 ° C. for 1 hour with a final volume of 50 μL yielded dedecapped RNA. To the sample, 20 units of T4 polynucleotide kinase (PNK) was added to 10 μL of 10 μM ATP, 10 μM PTP reaction buffer (700 mM Tris-Cl, 100 mM MgCl ₂ , and 50 mM DTT, pH7.6) and the final volume was 100 μL at 37 ° C. Incubate for 30 minutes at. As a result, the phosphate group was attached to 5'-OH, and the 3'-phosphate group was removed to obtain RNA having 5'-monophosphate and 3'-OH.

(3) self linking reaction

300 ng of RNA with 5'-monophosphate and 3'-OH from the trimming reaction was added to ₂ μL of 10 × CircLigase II buffer (0.33 M Tris-acetate pH7.5, 0.66 M potassium acetate, 5 mM DTT), and 50 μM MnCl ₂ . 5M betaine was added and the final volume was incubated at 60 ° C. for 1 hour. As a result, the 5'-monophosphate end and the 3'-OH end of the mRNA were linked to form a circular RNA.

In the above experiments, 300 ng of the trimming reaction product was recognized by the 5'-monophosphate, XRN1 enzyme 2unit (NEB) with 5 '->3' excoribonuclease activity, 10x XRN-1 reaction buffer (1M NaCl, 500 mM Tris ₂ μL-Cl, 100 mM MgCl ₂ , 10 mM DTT) was added followed by incubation at 37 ° C. for 1 hour with a final volume of 20 μL. Electrophoresis of the incubation product revealed no band detected, indicating that all RNA was degraded. In other words, the 5 'end of the fragmented UHRR was all converted to monophosphate. In addition, 300ng of the trimming reaction product recognized 3'-OH RNaseR enzyme 2unit (Epicentre), 10x RNaseR reaction buffer (0.2M Tris-Cl pH 8.0, 1M KCl with 3 '->5' excoribonuclease activity) ₂ μL of 0.1 mM MgCl ₂ ) was added and the final volume was incubated at 37 ° C. for 1 hour. Electrophoresis of the incubation product revealed no band detected, indicating that all RNA was degraded. That is, the 3 'end of the fragmented UHRR was all converted to OH.

In addition, 300 ng of the self-linking reaction product recognizes the 3'-OH of the linear RNA, which has the 3 '-> 5' excoribonuclease activity and the presence of the RNaseR enzyme that does not degrade the linear RNA without the 3'-OH group. Incubated at 37 ° C. for 1 h. Electrophoresis of the incubation product revealed a band, indicating that fragmented UHRR was self-linked by RNA ligase.

9 is a photograph showing the results of electrophoresis of the product obtained by incubating the trimming reaction product and the self-linked product in the presence of XRN1 and RNaseR, respectively. In FIG. 9, Ladder represents the size marker, Intact UHRR represents the unirradiated UHRR, Fragmented UHRR represents the ultrasonically irradiated UHRR, and Trimming represents the trimming reaction product in the absence (-) of XRN1 and RNaseR, and the presence of XRN1. (XRN1), or results of incubation in the presence of RNaseR (RNaseR), and Ligation represents the result of incubation of the self-linked reaction product in the absence (-) or presence of RNaseR (RNaseR).

(4.1) Water-in-oil type Drop Formation and Polymerization

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. An activator mixture was obtained. Next, 400 μl of the oil-surfactant mixture was transferred to a cryotube vial and mixed with stirring at 1000 rpm for 5 minutes using a 3 × 8 mm stub.

Aqueous phase with 2 ng self-linked UHRR (40 mM Tris-HCl pH 8.8, 6.4 mM (NH ₄ ) ₂ SO ₄ , 53 mM KCl, 10.6 mM MgSO ₄ , 0.1% Triton X-100, 30 μM random hexamer, 1.33 mM dNTPs, 4.8 unit / μl Bst DNA polymerase, 16 unit / μl M-MLV reverse-trancriptase, 40 ng / μL T4 SSB and 0.2% Antifoam) 200 μl into a frozen tube vial containing the oil-surfactant mixture Was added dropwise 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%). The droplet together with the circular RNA, reverse transcription polymerase, DNA polymerase together with the components necessary for the polymerization reaction, the amplification reaction can be carried out in the droplet. Prototype RNA is sufficiently diluted to contain at most 50% of the total number of droplets.

(4.2) Polymerization

The emulsions were incubated at 45 ° C. for 15 hours each to allow reverse transcription and DNA dependent DNA polymerization. After the reaction, RNase was added and incubated to degrade the remaining RNA.

After completion of the reaction, the emulsion was centrifuged at 13,000 g for 5 minutes to remove the oil phase. 1 ml of diethyl ether was added to the aqueous phase to break up the droplets of the emulsion and remove the mineral oil.

(5) qPCR Correlation before and after amplification using correlation ) Confirm

The proportion of genes after amplification in the emulsion using the linear RNA and the self-linked reaction product of the fragmented UHRR was determined to confirm that the transcript amplification in the emulsion using the circular RNA was performed without bias.

Five reference genes were selected for this purpose. Five standard genes are beta-Actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-glucuronidase B (beta-glucuronidase). : GUSB), ribosomal protein, large PO (ribosomal protein, large, P0: RPLP0), transferrin receptor p90, and transferrin receptor p90 (CD71: TFRC) genes. RNA circularized by self-linkage was introduced and amplified in droplets in the emulsion according to (4.1) and (4.2).

Primer-probe sets that specifically bind to these five genes were selected and DNA was quantified by qRT-PCR of the fragmented UHRR linear RNA as a template (hereinafter referred to as "amplification product from linear RNA in sample"). . Next, the linear RNA of fragmented UHRR was circularized according to (1), (2), (3), (4.1) and (4.2), and 2ng of the circularized RNA was used as a template and introduced into the droplets in the emulsion. Amplification by RT-RCA. As a result, 21 μg of amplification product was obtained. DNA was quantified by qPCR using this amplified product as a template and a primer-probe set that specifically binds to five genes (hereinafter also referred to as "amplification product from circular RNA"). By comparing the amplification product from the linear RNA and the amplification product from the circular RNA in the sample, the correlation between the original RNA amount and the DNA amount corresponding to the RNA amplified by amplification in droplets in the emulsion in the circularized RMA was confirmed. Table 2 shows the primer-probe sets used.

Target genes designation SEQ ID NO: Beta-actin B-actin_F 11 B-actin_R 12 B-actin_P 13 GAPDH GAPDH_F 14 GAPDH_R 15 GAPDH_P 16 GUSB GUSB_F 17 GUSB_R 18 GUSB_P 19 RPLPO RPLPO_F 20 RPLPO_R 21 RPLPO_P 22 TFRC TFRC_F 23 TFRC_R 24 TFRC_P 25

Table 3 shows the amplification products from the linear RNA and the amplification products from the circular RNA in the samples for the five RNAs, i.e. the amount of DNA corresponding to the RNA amplified by the amplification in the droplets in the emulsion in the original RNA amount and the circularized RNA Pearson correlation coefficient.

Target genes QRT-PCR product templated with UHRR (RNA) * QPCR product (DNA) based on emulsion amplification product (DNA) ** Actin 3.9 x 10 ⁶ 5.8 x ^{10 8} GAPDH 3.3 x 10 ⁶ 1.9 x ^{10 8} RPLPO 8.2 x 10 ⁵ 1.2 x ^{10 8} TFRC 6.1x10 ⁴ 1.5 x 10 ⁷ GUSB 1.5 x 10 ⁴ 1.1 x 10 ⁷ Pearson's Correlation Coefficient 0.87

As shown in Table 3, it was confirmed that the Pearson correlation coefficient was 0.87 to generate DNA from the target RNA so as to have a high similarity with the ratio of the plurality of RNAs initially present.

<110> Samsung Electronics Co., Ltd. <120> Method amplifying DNA from RNA in a sample <130> PN096288 <160> 25 <170> KopatentIn 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 ccactttgcc tttctctcca 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 cattcatttg tttacatctg gc 22 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> template specific primer <400> 3 ggaaacggat gataactggt 20 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> cDNA binding primer <400> 4 acattcattt gtttacatct ggc 23 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> downstream primer for actin <400> 5 gtcatagtcc gcctagaagc 20 <210> 6 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> downstream primer for GUSB <400> 6 gccctgactc ggggagg 17 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> downstream primer for TFRC <400> 7 cagccactgt aaactcaggc c 21 <210> 8 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> upstream primer for actin <400> 8 gaaattaata cgactcacta tacctggcct cgctgtccac 40 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> upstream primer for GUSB <400> 9 gaaattaata cgactcacta taccaggtat ccccactcag 40 <210> 10 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> upstream primer for TFRC <400> 10 gaaattaata cgactcacta tacctggact atgagaggta c 41 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> B-actin_F <400> 11 cagcagatgt ggatcagcaa g 21 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> B-actin_R <400> 12 gcatttgcgg tggacgat 18 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> B-actin_P <400> 13 aggagtatga cgagtccggc ccc 23 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_F <400> 14 attccaccca tggcaaattc 20 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_R <400> 15 gatgggattt ccattgatga ca 22 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_P <400> 16 ccgttctcag ccttgacggt gc 22 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GUSB_F <400> 17 cccactcagt agccaagtca 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GUSB_R <400> 18 cacgcaggtg gtatcagtct 20 <210> 19 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GUSB_P <400> 19 tcaagtaaac gggctgtttt ccaaaca 27 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> RPLPO_F <400> 20 ccattctatc atcaacgggt acaa 24 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> RPLPO_R <400> 21 tcagcaagtg ggaaggtgta atc 23 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> RPLPO_P <400> 22 tctccacaga caaggccagg actcg 25 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TFRC_F <400> 23 gccaactgct ttcatttgtg 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TFRC_R <400> 24 actcaggccc atttccttta 20 <210> 25 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> TFRC_P <400> 25 agggatctga accaatacag agcagaca 28

Claims

a) providing a sample comprising a plurality of types of RNA;
b) incubating the sample in the presence of the following enzyme to produce a mixture comprising at least two RNAs having 5′-monophosphate and 3′-OH in the sample: 5′-cap structure to 5′-monophosphate An enzyme converting RNA having a 5'-cap structure in the sample to an RNA having 5'-monophosphate, an enzyme phosphorylating 5'-OH as an enzyme to convert 5 An enzyme for converting the phosphorylated 3'-terminus into an RNA having 3'-OH in the sample as an enzyme for converting to an RNA having a '-monophosphate;
c) incubating the mixture after step b) in the presence of RNA ligase to generate circular RNA from two or more RNAs having the 5'-monophosphate and 3'-OH, wherein the RNA ligase is -Catalyzing each intramolecular linkage of at least two RNAs with monophosphate and 3'-OH;
d) incubating the circular RNA in the presence of RNA-dependent DNA polymerase to generate DNA from the circular RNA;
e) identifying the sequence of the DNA molecule corresponding to the sequence of two or more RNAs having the 5'-monophosphate and 3'-OH in the sample from the generated DNA and the 5'-monophosphate and 3 in the sample Determining the amount of DNA molecules corresponding to two or more RNAs having '-OH; And
f) determining the proportion of the amount of DNA molecules corresponding to two or more RNAs having 5'-monophosphate and 3'-OH in the sample, wherein the proportion of the amount of the DNA molecule is the 5'- in the sample Correlating with a ratio of the amount of at least two RNAs with monophosphate and 3'-OH.

The method of claim 1, wherein 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'-OH and 3'-OH; RNA with 5'-monophosphate and 3'-OH; And RNA having 5'-monophosphate and 3'-monophosphate.

The method of claim 1, wherein the sample comprises mRNA isolated from a biological sample.

The method of claim 1, wherein the sample comprises a degraded product of RNA isolated from a biological sample.

The method of claim 1, wherein the sample comprises RNA isolated from a formalin-fixed paraffin-embedded (FFPE) tissue sample.

The method of claim 1, wherein the enzyme converting the 5′-cap structure to 5′-monophosphate is tobacco acid pyrophosphatase (TAP), DCP, or a combination thereof.

The method of claim 1, wherein the enzyme phosphorylating 5′-OH is T4 polynucleotide kinase (PNK).

The method of claim 1, wherein the enzyme that dephosphorylates the phosphorylated 3′-end is T4 polynucleotide kinase (PNK).

The method of claim 1, wherein the RNA-dependent DNA polymerase has strand substituted DNA polymerase activity.

The method of claim 1, wherein the RNA-dependent DNA polymerase is Bst DNA polymerase.

The method of claim 1, wherein step d) is performed in the presence of a random primer.

The method of claim 1, further comprising incubating the sample in the presence of poly (adenylate) polymerase prior to step c) to add a polyadenylate sequence to the 3′-end.

The method of claim 12, wherein step d) is performed in the presence of an oligo dT primer.

The method of claim 1, further comprising forming a water-in-oil droplet comprising, after step d), the circular RNA and the elements required for nucleic acid polymerization.

The method of claim 1, wherein the elements required for nucleic acid polymerization include reverse transcriptase, DNA-dependent DNA polymerase, or a combination thereof.

The method of claim 14, wherein step d) is performed at isothermal.

The method of claim 14, wherein step d) is performed at 40 to 50 ° C. without thermocycling.

The method of claim 1, wherein step d) is incubating in the presence of a DNA-dependent DNA polymerase in addition to an RNA-dependent DNA polymerase.