KR20160127517A - Methods for Simultaneous Detecting Plurality of miRNAs - Google Patents
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Abstract
The present invention relates to a method for simultaneously quantitatively detecting a plurality of miRNAs in high specificity. By using the method of the present invention, quantitative detection of more than 50 kinds of miRNAs is possible at the same time.
Description
The present invention relates to a method for simultaneously quantitatively detecting a plurality of miRNAs in high specificity.
miRNAs are small noncoding RNAs of 18-23 nucleotides, with the following developmental steps: i) transcription by RNA polymerase II from intracellular genes. ii) 80-150 nucleotides (nts) The form of primary miRNA (pri-miRNA) is mainly derived from DNA between the intron part of the pre-mRNA or between the gene and the gene. iii) 60-100 nt of pre-miRNA is formed by Drosha-DGCR8 in the nucleus. iv) Pre-miRNA is exported to cytoplasm by Exportin5-RanGTP. v) Pre-miRNA becomes miRNA (mature miRNA) by Dicer-TRBP. vi) mature miRNA binds to the 3 'untranslated region (3'-UTR) of the target messenger RNA (mRNA) along with RNA induced silencing complex (RISC) to degrade mRNA and inhibit protein synthesis.
miRNAs are known to be involved in a variety of biological processes, namely cell growth, differentiation, and development, and are used as biomarkers in biological research.
Over the last seven to eight years, a large number of miRNA databases have been created (miRBase, microRNA, org, miRGen 2.0, etc.) and more than 1800 human miRNAs have been registered and total miRNAs reach 28,000 (miRBase 21). Currently, the role of each miRNA in cell / biotissue research is actively underway at home and abroad. Especially, the pattern change of miRNA expression profile is closely related to the specific state of disease, so quantitative research There is a surge in interest.
Because one miRNA has a large number of putative target mRNAs and a number of miRNAs are involved in one disease system, it is fast and accurate to quantitatively observe several miRNAs and their target mRNAs, Highly multiplexed analytical techniques are required for a large number of RNAs.
As research on miRNAs develops and the number of registered miRNAs in a database grows, there is a growing demand for efficient analysis methods that can measure multiple miRNAs and target mRNAs simultaneously with high quantitative specificity and rapidity. No appropriate technical alternative has been reported.
In addition, qualitative and quantitative analysis of miRNAs can be more challenging due to the specificity of miRNAs. First, the length of mature miRNA (hereinafter referred to as miRNA) is 23 bp (base-pair) or less, which is much shorter than that of mRNA. When the miRNA sequence is compared with the target transcript or pri- . This currently reduces quantitative specificity in RT-qPCRs that are mainly used for miRNA quantification. Also, the ratio of GC base varies in a large range, which makes it difficult to determine optimal reaction conditions in the PCR reaction and leads to inaccuracies such as low reproducibility of the assay results. In particular, miRNAs with very similar nucleotide sequences are highly likely to co-amplify by non-specific annealing with the primers during PCR, and these non-specific amplifications ) Are required to be excluded from the analysis results.
In order to overcome these technical limitations, the present invention proposes a method for quantifying more than 50 miRNAs and target mRNAs in a short time, more accurately than the conventional techniques, and quantifies miRNAs used as biomarkers, .
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
The present inventors have made extensive efforts to develop a method capable of quantitatively detecting a plurality of miRNAs simultaneously at a high specificity. As a result, SBE reaction was performed using the cDNA sequence of the miRNA to be measured and a competitor sequence thereof, and the resulting product was analyzed by mass spectrometry to determine that a large number of miRNAs could be quantitatively detected simultaneously. It was completed.
Accordingly, it is an object of the present invention to provide a method for quantitative detection of miRNA.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
According to one aspect of the present invention, the present invention provides a miRNA quantitative detection method comprising the steps of:
(a) preparing a sample of interest for quantitative detection of miRNA;
(b) generating cDNA for the miRNA through RT-PCR on the target sample;
(c) adding a competitor sequence to the cDNA to a target sample at a reference concentration and performing PCR amplification;
(d) adding a single base extension (SBE) primer and ddNTP to the target sample and performing an SBE reaction; And
(e) measuring the ratio of the content of the extension product to the cDNA and competitor sequence, and thereby calculating the miRNA concentration in the sample.
The present inventors have made extensive efforts to develop a method capable of quantitatively detecting a plurality of miRNAs simultaneously at a high specificity. As a result, it was confirmed that many miRNAs can be quantitatively detected at the same time by SBE reaction using the cDNA sequence of the miRNA to be measured and a competitor sequence thereof, and by analyzing the resulting product by mass spectrometry.
The miRNA of the present invention is a small noncoding RNA consisting of 18-23 nucleotides and is known to be involved in various biological processes such as cell growth and differentiation and development, It has been known as a biomarker. It has been found that pattern changes in the expression profile of miRNAs are closely related to the specific state of the disease, and it is required to simultaneously measure the expression profiles of a large number of miRNAs. When using the detection method of the present invention, more than 50 kinds of miRNAs can be quantitatively detected with high specificity in a single trial.
Hereinafter, each step of the quantitative detection method of miRNA of the present invention will be described in detail.
(a) preparing a target sample for quantitative detection of miRNA
The target sample of the present invention refers to a sample expected to contain a miRNA to be measured, and may be a biological sample separated from a living body, and specifically, for example, a cultured cell; From the brain, eye, heart, intestine, kidney, liver, lung, muscle, spleen or testis. Tissue obtained; Blood; Plasma; Serum; Urine; Saliva; Sweat; Semen; Or mucus. The detection method of the present invention can detect the sample using a very small amount of the sample.
In one embodiment of the invention, the subject sample of step (a) of the present invention comprises more than ten miRNAs. According to the method of the present invention, it is possible to simultaneously quantitatively detect many kinds of miRNAs at a higher efficiency and more accurately than by conventional methods. The target sample of step (a) of the present invention is capable of containing approximately ten miRNAs, which are limitations of the conventional methods, and it is further possible to quantitatively detect more than 50 miRNAs simultaneously.
(b) generating cDNA for the miRNA through RT-PCR on the target sample
The step of generating cDNA of miRNA to be detected by RT-PCR using the sample prepared in step (a). CDNA can be generated using a primer for reverse transcription of a miRNA to be detected, and primers for a plurality of miRNAs are simultaneously processed to simultaneously generate cDNAs for respective miRNAs. Primers for miRNAs to be detected can be readily selected by those of ordinary skill in the art through conventionally known information.
In one embodiment of the present invention, the RT-PCR of step (b) of the present invention is carried out using at least one primer selected from the group consisting of stem loop primer, flap primer and LNA primer. CDNA of about 60-100 bp in length can be generated using a stem loop primer (Chen et al., 2005 Nucleic Acids Research) used in the reverse transcription of the present invention (Ding and Cantor, 2003 PNAS; Diffield et al. 2010 The RNA Journal). The term "flap loop primer " of the present invention means a primer having a flap structure (Afonina et al. 2007 BioTechniques). Like the stem loop primer, a part of the miRNA to be analyzed, Terminus, and the portion of the flap is about 25-40 bp in length and may have a single nucleotide sequence or a random nucleotide sequence. As used herein, "LNA (locked nucleic acid) primer" (Koshkin et al., 1998 Tetrahedron) means a primer containing several nucleotides of the LNA structure and may include a flap structure. LNA primers can be annealed to miRNAs at significantly higher specificities, which has the advantage of improving the specificity of the assay. The primer of the present invention can be used without limitation as long as it is a primer capable of producing cDNA by specifically annealing to the nucleotide sequence of the miRNA to be analyzed in addition to the above-mentioned primer.
In one embodiment of the invention, the cDNA of step (b) of the present invention has a length of 30 bp to 150 bp. It is an easy length for detection of the mass ratio to the competitor sequence, more preferably 30-120 bp, even more preferably 40-120 bp, even more preferably 40-100 bp, still more preferably 50-100 bp, and still more preferably about 60-100 bp cDNA can be used.
(c) adding a competitor sequence to the cDNA to the target sample at a reference concentration, and performing PCR amplification
The competitor sequence for the cDNA generated in step (b) is added to the target sample at an arbitrary reference concentration determined, and the cDNA and the competitor sequence are PCR-amplified together. The term "competitor" of the present invention means a nucleotide sequence in which any one of the bases constituting the cDNA sequence is modified to another base. The "reference concentration" in step (c) of the present invention is not particularly limited as a value that can be arbitrarily determined by the practitioner. The reference concentration of the present invention is intended to quantitatively measure the amount of cDNA by measuring the relative amount of competitor and cDNA, and the practitioner can be appropriately selected according to the scale of detection experiment.
The cDNA and competitor are PCR amplified with each dNTP, forward primer, and reverse primer in a single reaction tube. The PCR process can be carried out according to a conventionally known method, and there is no particular limitation. In one embodiment of the present invention, 40 cycles have been performed and are not necessarily limited thereto.
(d) adding the single base extension (SBE) primer and ddNTP to the target sample and performing the SBE reaction
The step of performing the SBE reaction using the PCR product in step (c) as a template for a single base extension (SBE) reaction. A " single base extension (SBE) reaction "of the present invention is a method for detecting deformation of a nucleotide at a specific site of a nucleic acid sequence, which is a reaction for extending an SBE primer with a single base complementary to a base at a nucleotide modification site (S. Kim, et al. 2002 Nucleic Acids Research 30 e85; S. Kim, et al. 2003
The SBE reaction described above extends the SBE primer, which is an oligonucleotide, to ddNTP, which is complementary to a single base site (nucleotide modification site) between the cDNA and the competitor. The term "extension product " of the present invention means a product in which the SBE primer is extended by one ddNTP. The elongation products are produced in the same ratio as the content ratio of cDNA and competitor. Therefore, by measuring the content ratio of the two elongation products, the content ratio of cDNA and competitor can be measured. When 50 kinds of miRNAs are detected simultaneously, 50 kinds of SBE primers for each miRNA cDNA and 50 kinds of SBE primers for competitors can be simultaneously treated in one reaction tube.
In one embodiment of the invention, the ddNTP of step (d) of the present invention is a biotinylated ddNTP and further comprises the following step (d-1) between step (d) and step :
(d-1) contacting the resultant of step (d) with a biotin-binding protein coated stationary phase to purify the elongate product.
"Biotinylated ddNTP" of the invention refers to a ddNTP labeled with biotin, and the "biotin" do not affect the activity of the protein as a vitamin B 7 having a small size, a predetermined biotin-binding protein and the strong And can be used for biological analysis techniques. Biotin-binding protein-coated immobilized phases can be used to purify only bound biotinylated ddNTPs. The biotin-binding protein-coated "fixed phase" of the present invention refers to a support which is immobilized so that it is not removed during washing of residues not participating in the reaction, and specifically, for example, beads or plate- A solid support may be used, but is not limited thereto. The above-described fixed phase is coated with a biotin-binding protein, and only the extended product can be separated-purified by bringing the result of step (d) into contact with the stationary phase. This makes it possible to detect quantitative and specific miRNAs.
According to the prior art, mass spectra of remnants of the elongation reaction remain complex during mass spectrometry, which inhibits quantitative analysis and reduces the number of mRNAs that can be analyzed at one time. However, according to the present invention, which can separate and purify only the prolonged product, the accuracy of mass spectrometry can be drastically improved, and the number of mRNAs that can be assayed at the same time can be dramatically increased from about 10 to over 50 in the prior art.
In another embodiment of the present invention, the ddNTP of step (d) of the present invention is a mass-tagged ddNTP comprising a photodegradation linker (Bai et al. 2004 Nucleic Acids Research), wherein the SBE primer (D-2) and (d-3) between the step (d) and the step (e), wherein the measurement of the content ratio of step (e) By mass ratio of the mass-tag:
(d-2) contacting the resultant of step (d) with a biotin-binding protein coated stationary phase to purify the elongate product;
(d-3) separating the mass label by photo-decomposing the elongated product purified by the step (d-2).
In this embodiment using mass labeling, when quantitative detection of a plurality of miRNAs is performed, step (d) of the present invention is each performed in a separate reaction tube, step after step (d-2) ) Can be conveniently collected into one reaction tube. Specifically, for example, when quantitatively detecting miRNA1, miRNA2 and miRNA3, the cDNA of miRNA1 and its competitor, and a pair of SBE primers and the mass labeled ddNTP1, ddNTP2 for the cDNA and competitor are reacted in a single tube; a cDNA of miRNA2 and its competitor, and a pair of SBE primers and mass labeled ddNTP3, ddNTP4 for said cDNA and competitor are reacted in different tubes; The cDNA of miRNA3 and its competitors, and a pair of SBE primers and mass labeled ddNTP5, ddNTP6 for the cDNA and competitor can be reacted in another tube. The tube may preferably be a well-plate containing an appropriate number of tubes to take into account the number of miRNAs to be detected. After performing step (d), the reaction products contained in the tube may be combined into one reaction tube to perform steps (d-1), (d-2) and (e).
The mass label of the present invention can be used without restriction as long as it does not inhibit the SBE reaction. Specifically, for example, oligonucleotides, oligopeptides, oligosaccharides, various other organic substances, (Thomson et al., 2007 Nucleic Acid Research). The mass labeling assay method can improve the accuracy of quantitative analysis compared to the mass analysis of whole elongation products. Mass labeling allows the quantitative ratio of cDNA and its competitor to specific miRNAs to be quantitatively detected, thereby enabling quantitative detection of miRNAs. Specifically, for example, when an oligopeptide is used as a mass marker, it is possible to produce an amino acid having various masses indefinitely by increasing the number of amino acids contained in the oligopeptide, and more than 100 The mass labeling can be easily prepared.
In one embodiment of the invention, the biotin-binding protein described above is selected from the group consisting of avidin, streptavidin, NeutrAvidin, captavidin and tamavidin (Takakura Y et al. , Tamavidins-novel avidin-like biotin-binding proteins from the Tamogitake mushroom, FEBS J, 276 (5): 1383-97 (2009)).
(e) measuring the ratio of the content of the extension produnt to the cDNA and competitor sequence, and thereby calculating the miRNA concentration in the sample
In the step (d), the elongation product generated from the cDNA and the competitor as a template by the SBE reaction is generated with the same content ratio as the content ratio of the cDNA and the competitor sequence. Specifically, for example, an extension product of a cDNA extending to a template of cytosine (C) will contain guanine (G), and an extension product of a competitor's guanine (G) will be. The concentration of miRNA in the sample can be calculated by measuring the ratio of the two elongation products. The concentration of miRNA in the sample can be calculated by measuring the ratio of the elongation product to the principle that the initial mixing ratio of the cDNA and the competitor is maintained after the PCR amplification and the extension product is generated by the SBE reaction according to the maintained mixing ratio Based.
In one embodiment of the invention, the determination of the content ratio of step (e) of the present invention is by mass spectrometry. In the above-described embodiment using biotinylated ddNTPs, due to the difference in one base sequence between the cDNA and the competitor, the resulting extension sequence also shows a difference in one base sequence, so that a small difference in mass . Mass ratio can be used to measure the content ratio between extension sequences showing small differences in mass. In addition, since there is a difference in the kinds of SBE primers used for each miRNA, a gap between mass analysis peaks is generated, and it becomes possible to simultaneously measure the content ratios of two or more miRNAs. On the other hand, in the case of another embodiment of the present invention using the mass-labeled ddNTP and the biotinylated SBE primer comprising the photodegradation linker, the measurement of the content ratio of step (e) And the mass ratio of the mass-tag.
The mass spectrometry of the present invention can be freely used without any particular limitation, and specifically, for example, MALDI-TOF (matrix-assisted laser desorption / ionization time-of-flight), electro-spray ionization (ESI) , Quadrupole Ion Trap, or chemical ionization (CI).
The features and advantages of the present invention are summarized as follows:
(a) The present invention provides a miRNA quantitative detection method.
(b) Using the method of the present invention, quantitative detection of more than 50 kinds of miRNAs at the same time is possible.
Figure 1 shows an overview of miRNA assays using modified nucleotides.
Figure 2 shows the results of quantitative analysis of miR-486 using biotinylated ddNTP. When the concentration of the competitor is decreased by 10 times (from the left to the right), it is observed that the competitor peak is decreased by the corresponding amount, and it is confirmed that the quantitative analysis is possible.
FIG. 3 shows the results of quantitative analysis of three miRNAs (miR-29a, -29b, -29c) having similar nucleotide sequences using biotinylated ddNTPs. Different mass / charge peaks were observed for the three miRNAs, and no other peaks were found, demonstrating the specificity of the reaction.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
Example
Example 1: Demonstration of quantitative measurement capability
The amount of miRNA-486 was measured in the brain tissue of mice using biotinylated ddNTPs (see Figure 2). The miRNA was extracted from mouse brain tissue using mir Vana ™ and reverse transcribed using Ambion RETROScript reverse transcription kit. A 10 pmol stem loop primer (5'-GTCGTATCCA GTGCAGGGTC CGAGGTATTC GCACTGGATA CGACC TCGGG-3 ') To make the cDNAs longer than the original miRNAs. A competitor was a chemically synthesized oligonucleotide of the same base sequence as cDNA and a base was converted from C (cDNA) to G (competitor). The cDNA was then PCR amplified together with competitor in one reaction tube. For the PCR reaction, 3 nmol of each dNTP, 5 nmol (forward: 5'-TGGCATCCTG TACTGAGCT-3 '; reverse: 5`-GTGCAGGG TCCGAGGT-3') of a forward primer and a reverse primer, 2 μl of the reaction product and 2 units of Amplitaq GOLD Taq Polymerase were used. Thermal cycling was performed at 94 ° C for 3 minutes, at 94 ° C for 30 seconds, at -60 ° C for 30 seconds, then at 72 ° C for 30 seconds (repeated 40 times) , And 72 ° C for 5 minutes. At this time, the PCR product of the cDNA and the competitor all corresponded to each other except for one base and the same length. The PCR product was used as a template for the SBE reaction after treatment with Exonuclease I and Alkaline Phosphatase. 5 pmol of primer (5'-GGCATCCTGT ACTGAGCT-3 ', 18 mer) was reacted with 25 pmol of biotin-ddCTP or biotin-ddGTP and sequenced by a Thermo Sequenase (1 unit) site). < / RTI > The reaction product was separated and purified from other reaction materials using an extension product (19 mer oligonucleotide) to which C or G was added to the primer using an avidin-coated bead. The purified elongation product was analyzed by MALDI-TOF mass spectrometry (see Figure 2). As a result, two peaks were observed in the mass spectrum, each of which had a peak at which C was added to the primer (the template base was G, meaning that it was a peer derived from a competitor) and a peak added with G (the template base was C And thus it can be seen that it is a peak derived from the cDNA). The mass / charge value was confirmed. That is,
Example 2: Demonstration of high specificity for miRNA
(MiR-29a, -29b, -29c) that are very similar in nucleotide sequence (see FIG. 3). miRNAs were extracted from primary rat hepatocytes and reverse transcribed using Stem Loop RT primers. The stem loop RT primer sequences are as follows: 5'-GTCGTATCCA GTGCAGGGTC CGAGGTATTC GCACTGGATA CGACTAACCG-3 '(29a), 5'-GTCGTATCCA GTGCAGGGTC CGAGGTATTC GCACTGGATA CGACAA CACT-3' (29b), 5'- GTCGTATCCA GTGCAGGGTC CGAGGTATTC GCACTGGATA CGACTAACCG -3 '(29c).
After the RT reaction, the cDNA was PCR simultaneously with the competitor, and the competitor used one of the bases converted from C (cDNA) to G (competitor).
3 '(29a), 5'-GCGCTAGCAC CATTTGAAAT-3' (29b), 5'-GCGCTAGCAC CATTTGAAAT-3 '(29c) and reverse primers were 5'-GTGCAGGGTC CGAGGT -3 'was used. The primers used in SBE after PCR reaction were 5'-ACCATCTGAAA TCGGTTA-3 '(29a), 5'-GCACCATTTG AAATCAGTGTT-3' (29b), 5'-CACCATTTGA AATCGGTTAGTC-3 '(29c). As shown in FIG. 3, the C extension product (6147 Da) and the G extension product (6186 Da) of the SBE primer were found in the mass spectrum and no other unintended product was found. This means that when miRNAs are analyzed by the proposed method, other miRNAs with similar nucleotide sequences participate in the reaction and do not produce unwanted SBE products. Among the two peaks of the mass spectrum, the peak corresponding to the G extension product (6186 Da) is represented by the base sequence of the cDNA, and the peak corresponding to the C extension product (6147 Da) is represented by the competitor because the base sequence is G. The concentration of miRNA in the sample could be calculated from the relative ratio of the two peak areas. This result was confirmed independently using RT-qPCR, and it was confirmed that the miRNA to be studied can be specifically analyzed by the SPC-SBE and MALDI-TOF mass spectrometry (see FIG. 3) .
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (8)
(a) preparing a sample of interest for quantitative detection of miRNA;
(b) generating cDNA for the miRNA through RT-PCR on the target sample;
(c) adding a competitor sequence to the cDNA to a target sample at a reference concentration and performing PCR amplification;
(d) adding a single base extension (SBE) primer and ddNTP to the target sample and performing an SBE reaction; And
(e) measuring the ratio of the content of the extension product to the cDNA and competitor sequence, and thereby calculating the miRNA concentration in the sample.
(d-1) contacting the resultant of step (d) with a biotin-binding protein coated stationary phase to purify the elongate product.
(d-2) contacting the resultant of step (d) with a biotin-binding protein coated stationary phase to purify the elongate product;
(d-3) separating the mass label by photo-decomposing the elongated product purified by the step (d-2).
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