KR101822609B1 - Methods and kits for expression profiling of microRNA using PNA mediated Real-Time PCR clamping - Google Patents

Abstract

The present invention relates to a method and kit for analyzing the microRNA (miRNA) expression pattern using PNA-based real-time PCR clamping, and the method of analyzing miRNA expression pattern according to the present invention can be used for cell proliferation, differentiation and death, And miRNAs that have a similar base sequence can be used to identify miRNA subtypes that are important for the expression of miRNAs and specific mechanisms.

Description

[0002] Methods and kits for the expression of microRNA using PNA-based real-time PCR clamping [

More particularly, the present invention relates to a method and kit for analyzing microRNA expression patterns using PNA-based real-time PCR clamping, and more particularly, A method for analyzing the expression pattern of miRNA having a similar base sequence using the same, and a kit for use in the method.

MicroRNAs (miRNAs) are single-stranded RNA molecules of 21-25 nucleotides that control the expression of eukaryotic genes that bind to the mRNA 3 'untranslated region (UTR) of a particular gene, It is a regulatory substance that controls the translation process. In 1993, miRNAs were noticed in Caenorhabditis elegans in 1993, and some of these genes, let-7 and lin-4, were identified as small RNA fragments (non-coding RNA ). These RNAs were named small RNAs (stRNAs) because they were expressed at specific developmental stages and regulated their development. These miRNAs play a crucial role in the transient regulation of programs that regulate cellular development by inducing switch-off of target molecules. Hundreds of miRNAs have recently been identified and appear to regulate cell growth, differentiation and death in insects, flies and human genomes. More than 700 miRNAs have been identified in human genes (Nucleic Acids Res. 2006, 34: D140-144).

miRNA biosynthesis is initiated by transcription by RNA polymerase II and is produced in two steps: the first miRNA transcript (primary miRNA / pri-miRNA) is introduced into the nucleus by the RNase III type enzyme called Drosha (Pre-miRNA) with a stem-loop structure of about 70-90 nucleotides, and then the pre-miRNA is transferred to the cytoplasm and cleaved by an enzyme called Dicer to form 21 -25 nucleotides of mature microRNA (mature microRNA). The sequence of some miRNAs is highly conserved among species and is expected to be involved in important life phenomena. Until recently, many researchers have shown that miRNA plays an important role not only in cancer cells and stem cells, but also in cell proliferation, differentiation and death, and lipid metabolism.

However, many functions of miRNAs have yet to be elucidated, so research on miRNAs is actively under way. There are thousands of small RNA fragments in the cell. One of them is miRNA, which can be used to identify miRNAs that are closely related to specific diseases or cancers through the analysis of miRNA expression patterns, and the discovered miRNAs can be used as biomarkers to diagnose and predict disease (Seminars in cancer Biology 2008, 18: 89-102). Therefore, it is very important to analyze the expression pattern of miRNA.

A number of experimental techniques have been developed for the analysis of miRNA expression, and a microarray chip for analyzing the expression pattern of various miRNAs has been developed and used mainly in Northern blots (Nucleic Acids Res. 2003 , 31 (17): 4973-4980). A DNA chip is a chip in which DNA or PNA probes designed based on known genetic information on a solid surface are immobilized at a high density, and hybridization reaction with a target nucleic acid to be analyzed on a chip is detected by fluorescent labeling . The miRNA microarray technique can be used to analyze miRNAs specifically expressed from many cells or tissues at the same time. Therefore, it is useful for studying miRNA expression patterns since DNA chips can analyze various genetic information in a single experiment (Gynecologic Oncol. 2006, 100: 38-43; Nucleic Acids Res 2007, 35 (7): e48). The miRNA microarray technique described above is used to compare the expression pattern of various samples and to confirm the expression level and expression pattern of miRNA having a change in the expression pattern in the array for more accurate diagnosis, For sample analysis, microRNA real-time PCR technique has been widely used (Fertil Steril. 2008, 89 (6): 1771-1776).

Real-time PCR methods for miRNA expression analysis, which are currently in commercial use, usually use fluorescent reagents. Methods for measuring fluorescence largely use an intercalating method, a TaqMan ^占 LNA method, and a molecular beacon method (Nucleic Acids Res. 2007, 35 (19): e127; Nucleic Acids Symp Ser. 1997, 37: 255-256; Clin Chem Lab Med., 2003, 41 (4): 468-474.). A further set of procedures is required for miRNA characterization prior to using these methods. First, since miRNAs are short as 20-25 nt and can not be PCR by themselves, it is also necessary to increase the length of the miRNAs together with the reverse transcription to synthesize them with cDNA using an additional primer. (Nucleic Acids Res. 2005, 33 (20): e179). These two steps are performed separately in the same step.

In the case of dividing into two steps, the first step is to increase the length by chain reaction of the nucleotide sequence A with a polymerase at the 3 'end of the miRNA, and then the second step is to make cDNA using reverse transcriptase. Expression is analyzed by real-time PCR using forword primers and universal reverse primers for specific miRNAs with cDNA. When this method is used, fluorescence is measured using an intercalating method (Nucleic Acids Res. 2007, 35 (19): e127).

When complementing the length of miRNA and performing cDNA synthesis at the same stage, cDNA is synthesized using a different reverse primer for each specific miRNA. Since the reverse primer used in this case contains the loop structure of the pre-miRNA, it can complement the length of the miRNA and selectively synthesize only the cDNA of the specific miRNA. It is also different from the previous method using universal reverse primer that both forword primer and reverse primer used to amplify cDNA in real-time PCR process are specific to specific miRNA. Therefore, it is more specific than the universal reverse primer method. For this method, the fluorescence is measured using the TaqMan ^™ probe (Nucleic Acids Symp Ser. 1997, 37: 255-256).

These real-time PCR techniques are known to be the most efficient miRNA analysis and diagnostic method to date. However, in order to confirm miRNA expression patterns, miRNA subtype (single nucleotide different) or mutation of one nucleotide sequence (Nucleic Acids Res. 2008, 36 (5): e27).

On the other hand, PNA was first reported in 1991 as a pseudo-DNA in which a nucleic acid base is linked to a peptide bond rather than a phosphate bond (Science, 1991, 254: 1497-1500), synthesized by a chemical method, Hybridization reaction with the natural nucleic acid of the sequence to form a double strand. When the number of nucleic acid bases is the same, the PNA / DNA double strand is more stable than the DNA / DNA double strand, and the PNA / RNA double strand is more stable than the DNA / RNA double strand. The basic backbone of peptide nucleic acids is most often used in the case of N- (2-aminoethyl) glycine repeatedly linked by amide bonds as a basic skeleton of PNA. Unlike the basic structure of a negatively charged natural nucleic acid It is electrically neutral. The four nucleic acid bases present in PNA occupy a space similar to the nucleotide base of DNA and the distance between nucleotide bases is almost the same as that of natural nucleic acid. PNA is chemically more stable than natural nucleic acid, and biologically stable because it is not degraded by nuclease or protease. Since PNA is also electrically neutral, the stability of PNA / DNA, PNA / RNA double strands is not affected by salt concentration. Because of this property, PNAs are better able to recognize complementary nucleic acid sequences than natural nucleic acids and are therefore applicable for diagnostic or other biological and medical purposes.

The PNA clamping technique utilizes the advantages of the PNA described above, so that when the PNA probe is perfectly coupled, the enzyme does not recognize the amplification reaction and the PNA probe does not bind perfectly when there is a complementary sequence to the probe And thus the expression pattern of miRNA can be analyzed quickly and accurately by using the principle that the amplification reaction occurs.

Therefore, the present inventors have found that the miRNA can specifically bind to miRNAs 10a, 10b, 181a, 181b, 181c, 181d, 18a, 18b, 196a and 196b miRNAs in order to accurately analyze expression patterns of miRNA pseudo- And the expression pattern of the miRNA-like base sequence with high sensitivity and specificity can be accurately analyzed by using the PNA probe. Thus, the present invention has been completed.

It is an object of the present invention to provide a method for analyzing miRNA expression patterns using PNA-based real-time PCR clamping.

It is another object of the present invention to provide an assay kit for use in a method for analyzing expression pattern of miRNA using PNA-based real-time PCR clamping.

The first aspect of the present invention is

(1) synthesizing cDNA from total RNA or target microRNA (microRNA, miRNA) to be detected;

(2) a PNA (Peptide) which can bind the cDNA perfectly to a clamping primer set of a specific miRNA and a single nucleotide differentiation of the miRNA, and specifically binds to a region containing a different base sequence among subtypes of the miRNA Performing real-time polymerase chain reaction (PCR) on the miRNA in the presence of a Nucleic Acid clamping probe; And

(3) analyzing the result of the real-time PCR to confirm miRNA expression:

, ≪ / RTI > and methods for analyzing miRNA expression patterns.

The second side of the present invention

The present invention relates to a kit for use in a method for analyzing an miRNA expression pattern according to the present invention, which comprises any one of the PNA clamping probes selected from SEQ ID NOS: 1-64.

The PNA probe according to the present invention is very stable to biological enzymes and physical factors and is very simple to analyze and can be used for mass analysis and clinical use very easily since its expression is analyzed within a short time.

In addition, the method of analyzing the expression pattern of miRNA according to the present invention can be used to identify expression patterns and specific mechanisms of miRNAs that play important roles in cell proliferation, differentiation and death, lipid metabolism, By enabling classification, it can be useful for miRNA research or diagnosis.

FIG. 1 is a schematic diagram showing a PNA-based real-time PCR clamping principle using a PNA clamping probe for confirming miRNA expression patterns according to the present invention,
(A: Real-time PCR clamping principle based on PNA on cDNA, B: Real-time PCR clamping principle based on PNA in cDNA synthesis on miRNA)
FIG. 2 is a graph showing the results of the conventional methods for the subtypes of hsa-miR-18a, hsa-miR-10a, hsa-miR-10b, hsa-miR-196a and hsa-miR- △ C _t ).
Figure 3 is a graph comparing the detection specificity ([Delta] Ct) of the miRNA expression pattern using a PNA clamping probe according to the present invention.
FIG. 4 is a graph comparing miRNA expression according to the subtype concentration of hsa-miR-10a miRNA mixed with Target hsa-miR-10b against the conventional method and the miRNA expression pattern detection specificity (ΔC _t ) of the present invention,
FIG. 5 is a graph comparing the miRNA expression according to the subtype concentration of hsa-miR-10b miRNA mixed with Target hsa-miR-10a to the miRNA expression detection specificity (ΔC _t ) of the present invention,
FIG. 6 is a graph comparing the miRNA expression according to the subtype concentration of miRNA of hsa-miR-18a mixed with Target hsa-miR-18b to the miRNA expression detection specificity (ΔC _t ) of the present invention,
FIG. 7 is a graph comparing the miRNA expression according to the subtype concentration of miRNA of hsa-miR-18b mixed with Target hsa-miR-18a to the miRNA expression detection specificity (ΔC _t ) of the present invention,
FIG. 8 is a graph comparing the miRNA expression according to the subtype concentration of miRNA of hsa-miR-181a mixed with the target hsa-miR-181b to the miRNA expression detection specificity (ΔC _t ) of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention combines PNA clamping technology using a PNA probe and real-time PCR technology to analyze miRNA expression patterns more easily and easily.

1. PNA Clamping Design and manufacture of probes

The PNA probes of the present invention are each capable of perfectly binding to a subsequence having a miRNA-like base sequence, more than 10, preferably 13 to 30, more preferably 13 to 20, most preferably 14 to 20, And is composed of 22 nucleotide sequences.

The PNA probes of the present invention can be used in the present invention as well as in the case of HSA-miR-18a, hsa-miR-18b, hsa-miR-18b, hsa-miR- 181b, hsa-miR-181c, hsa-miR-181d, hsa-miR-196a and hsa-miR-196b. For example, the PNA probe of the present invention may comprise a nucleotide sequence of any one of SEQ ID NOS: 1 to 64 described in Table 1 below. PNA probe sequences within a range that can be easily modified by those skilled in the art from the above base sequence will be considered to be within the scope of the present invention. As a PNA probe having a length of 10mer or longer, Is included within the scope of the present invention as long as miRNA expression amount can be effectively comparatively analyzed using PNA-based real-time PCR clamping.

Specifically, SEQ ID NOS: 1 to 4 are designed to bind to the miRNA hsa-let7a subtype completely, and are designed to be able to distinguish the subtypes besides hsa-let7a, let7b, let7c, let7d, let7e, let7f, let7g and let7i And the region where the difference exists is included. SEQ ID NOS: 5 to 8 were designed to bind perfectly to the hsa-let7c subtype and also showed differences in nucleotide sequence so that they could be distinguished from let7a, let7b, let7d, let7e, let7f, let7g and let7i subtypes other than has-let7C . SEQ ID NOS: 9 to 12 are designed to bind perfectly to the hsa-let7g subtype, and the nucleotide sequence is different so as to be distinguishable from let7a, let7b, let7c, let7d, let7e, let7f and let7i subtypes other than has-let7g . SEQ ID NOS: 13 to 17 were designed to bind to the hsa-miR-10a subtype completely, and designed to include regions where nucleotide sequence differences occur so as to be distinguishable from hsa-miR-10b. SEQ ID NOS: 18-22 were designed to bind perfectly to the hsa-miR-10b subtype, and designed to include sites where differences in base sequence occur so as to be distinguishable from hsa-miR-10a. SEQ ID NOS: 23 to 29 were designed to bind to the hsa-miR-18a subtype completely, and designed to include sites where differences in base sequence occur so as to be distinguishable from hsa-miR-18b. SEQ ID NOS: 30 to 34 were designed to bind perfectly to the hsa-miR-18b subtype, and designed to include sites where differences in base sequence occur so as to be distinguishable from hsa-miR-18a. SEQ ID NOS: 35 to 39 were designed to perfectly bind to the hsa-miR-181a subtype, and the sites where differences in nucleotide sequences were generated so as to be distinguishable from hsa-miR-181b, hsa-miR-181c and hsa-miR- . SEQ ID NOs: 40 to 45 were designed to bind to the hsa-miR-181b subtype completely, and the region where the nucleotide sequence difference occurred so as to be distinguishable from hsa-miR-181a, hsa-miR-181c and hsa-miR- . SEQ ID NOS: 46 to 51 were designed to bind to the hsa-miR-181c subtype completely, and the site where the nucleotide sequence difference occurred so as to be distinguishable from hsa-miR-181a, hsa-miR-181b and hsa-miR- . SEQ ID NOS: 52-57 were designed to bind perfectly to the hsa-miR-181d subtype, and the sites where the nucleotide sequence difference occurred so as to be distinguishable from hsa-miR-181a, hsa-miR-181b and hsa-miR- . SEQ ID NOS: 58 to 61 were designed so as to bind to the hsa-miR-196a subtype completely, and designed to include a site where the nucleotide sequence difference occurs so as to be distinguishable from hsa-miR-196b. SEQ ID NOS: 62-64 were designed to bind perfectly to the hsa-miR-196b subtype and were designed to include sites where differences in nucleotide sequence could be distinguished from hsa-miR-196a.

The PNA probes of the present invention can include N-terminal or C-terminal hydrophilic functional groups to increase reaction efficiency and solubility, for example, at the N- or C-terminus (J Chem Technol Biotechnol. 2006, 81: 892-899; Tetrahedron Lett. 1998, 39: 7255-7258; Proc Natl Acad Sci USA 2002 , 99: 5953-5958; Anal Chem., 1997, 69: 5200-5202). As a specific example, a PNA probe with one lysine at the N-terminus was used.

The PNA oligomer used in the present invention is a PNA monomer protected with a Bts (Benzothiazolesulfonyl) group according to the method of Korean Patent No. 464,261 or a PNA monomer protected with a known Fmoc (9-flourenylmethloxycarbonyl) or t-Boc (t-butoxycarbonyl) (J Organic chem., 1994, 59 (19): 5767-5773; J Peptide Sci. 1995, 1 (3): 175-183).

2. Real - time for miRMA detection PCR Primer and cDNA Synthesis

In the present invention, the real-time PCR primer and cDNA synthesis for miRMA detection can be performed by conventional methods used for detection of miRNA.

Typically, it is possible to use primers and probes designed to synthesize cDNA from miRNA as a reverse primer in a loop structure and to detect the synthesized cDNA as a template with a forward primer and a reverse primer and a TaqMan ( ^TM) probe (Nucleic Acids Res. 2005, 33 (20): e 179).

In addition, A was continuously synthesized at the 3 'end of miRNA using Adenylation Polymerase, and cDNA was synthesized by using oligo dT primer and reverse transcriptase complementary to the synthesized A, and then synthesized cDNA was used as a forward primer and universal reverse primer (BioTechniq. 2005, 39 (4): 519-525).

In addition, a PNA (Peptide Nucleic Acid) clamping probe that can bind perfectly to a specific subtype of miRNA during cDNA synthesis and specifically binds to a region containing a base sequence that is different from other subtypes of the miRNA is further applied It is also possible to improve the specificity.

3. Real-time PCR based on PNA Detection of miRNA subtypes using clamping

The method for analyzing the expression pattern of miRNA subtypes according to the present invention

(1) synthesizing cDNA from total RNA or target microRNA (microRNA, miRNA) to be detected;

(2) a PNA (Peptide) which can bind the cDNA perfectly to a clamping primer set of a specific miRNA and a single nucleotide differentiation of the miRNA, and specifically binds to a region containing a different base sequence among subtypes of the miRNA Performing real-time polymerase chain reaction (PCR) on the miRNA in the presence of a Nucleic Acid clamping probe; And

(3) analyzing the result of the real-time PCR to confirm miRNA expression:

.

The total RNA or miRNA used in the step (1) of the present invention is extracted from the target sample and used. In the present invention, there is no particular limitation on the extraction of RNA. Any RNA extraction method generally used can be used. RNA extraction is carried out by extracting RNA from a sample to be expressed using a commercially available RNA extraction kit or the like.

In the present invention, the cDNA of step (1) is synthesized in the presence of a universal primer or a reverse primer which is a specific miRNA specific primer.

More specifically, in step (1), conventional methods used for detection of miRNA can be applied as a step of synthesizing cDNA using RNA extracted from a detection subject. A representative example is a method using a primer and a probe designed to synthesize cDNA from miRNA as a reverse primer of loop structure and to detect the synthesized cDNA as a template with a forward primer, a reverse primer and a TaqMan ^™ probe (Nucleic Acids Res. 2005, 33 20): A method for synthesizing cDNA from miRNA as a reverse primer of loop structure as disclosed in e179 is available.

As another example, A is continuously synthesized at the 3 'end of miRNA using Adenylation Polymerase, and synthesized cDNA is synthesized by using oligo dT primer and reverse transcriptase complementary to the synthesized A, and the synthesized cDNA is used as forward primer and universal reverse primer (BioTechniq., 2005, 39 (4): 519-525), and synthesized A-complementary oligo dT primer and reverse transcriptase A method of synthesizing cDNA by using the above-mentioned method is also available.

Also, in the present invention, the cDNA of step (1) may be a PNA (Peptide) which can bind to the subtype of the reverse transcription primer and the miRNA and specifically binds to a region containing a different base sequence among the miRNA subtypes Nucleic Acid) clamping probes.

In the present invention, in step (2), the cDNA synthesized in step (1) can be completely bound to a clamping primer set of a specific miRNA and a single nucleotide differentiation of a miRNA, and a base Performing real-time PCR on the miRNA in the presence of a PNA (Peptide Nucleic Acid) clamping probe that specifically binds to a region containing the sequence, wherein the various primers for miRNA detection are all Available.

In the present invention, the subtype of the miRNA is selected from the group consisting of has-let7a, has-let7c, has-let7g, hsa-miR-10a, miR-10b, hsa-miR- hsa-miR-181b, hsa-miR-181c, hsa-miR-181d, hsa-miR-196a and hsa-miR-196b.

In the present invention, the PNA clamping probe comprises a base sequence having a length of 13 to 30 mers, and more preferably, any one selected from SEQ ID NOS: 1-64.

In the present invention, in step (3), cDNA amplification by real-time PCR is analyzed to compare miRNA expression of two or more samples, and the presence or absence of miRNA expression or the expression level of miRNA can be compared with the amplified Ct value.

More specifically, when the PNA clamping probe of the present invention, which is designed to hybridize with miRNA sub-miRNAs, is hybridized to miRNA subtypes and inhibits miRNA amplification, the amplification is inhibited and the Ct value is high. If the specific miRNA to be expressed is PNA If the clamping probe is not fully hybridized to the miRNA subtype, the miRNA is amplified and the Ct value is low. Based on this principle, the PNA clamping probes of the present invention can compare the expression of more accurate miRNAs by suppressing nonspecific amplification according to pseudo-nucleotide sequences.

The real-time PCR method allows the quantitative analysis to be performed more precisely because the amount of the initial sample in which exponential amplification occurs is represented by the number of cycles in which the exponential increase of the fluorescent substance starts to be detected (Cycle threshold Can be analyzed in real time. This method is a method to quickly and easily diagnose the amplification product by omitting the step of measuring the intensity with the image analyzer by electrophoresis and automating and quantifying the amplification degree of the amplification product.

In the present invention, the analysis of the miRNA expression pattern is performed by one or more methods selected from the TaqMan probe method, the intercalating method, and the molecular beacon (Molecular Beacon) method.

For example, in the present invention, a TaqMan ^™ probe hybridized to a template is degraded by the 5 '→ 3' exonuclease activity of Taq DNA polymerase during an extension reaction, and the fluorescent dye is released from the probe, And the TaqMan probe method in which fluorescence was emitted was used.

The miRNA real-time PCR method using the PNA clamping probe of the present invention can be used when a precise comparison is difficult due to a subtype having a very similar base sequence. In addition to a study for comparing miRNA expression, a mechanism involved in the miRNA signaling system It can be used very usefully. It can also be effectively applied to studies that require large sample analyzes for specific miRNAs with subtypes.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not intended to limit the scope of the present invention in any way.

[ Example  One] miRNA  For confirmation of expression pattern PNA Clamping Probe  synthesis

PNA probes were synthesized to inhibit the amplification of other pseudomonucleotide sequences for the isolation of pseudomonucleotides such as miRNAs let7a, let7c, let7g, 10a, 10b, 181a, 181b, 181c, 181d, 18a, 18b, 196a, .

PNA clamping probes are used to detect miRNAs such as miRNAs let7a, let7c, let7g, Sixteen PNA probes were prepared as shown in Table 1 so as to be perfectly coupled to the probe probes 10a, 10b, 181a, 181b, 181c, 181d, 18a, 18b, 196a and 196b. As a probe that perfectly binds to each miRNA subtype, it is devised that the base sequence of the site where the base difference of the subtype is generated is located in the middle of the probe for the effective isolation of the subtype. PNA probes were synthesized according to the method described in Korean Patent No. 464261 (Org Lett. 2007, 9: 3291-3293).

[ Example  2] Standardized miRNA Synthesis of

In order to confirm the correct miRNA subtype resolution, synthetic RNAs of 21 to 25-mer length were synthesized with reference to biona (Korea) in the same manner as mature miRNA base sequences.

[ Example  3] miRNA  For subtyping miRNA  For the distinction of subtypes PNA The probe  Real time PCR Clamping  Establish method ( cDNA clamping ; 1)

(a) To 5 μl of synthetic miRNA (0, 0.8, 8, 80 pg / μl) prepared by concentration, add 3 μl of cDNA synthesis primer, 0.15 μl of dNTP mix (100 mM), 1 μl of reverse transcriptase, 1.5 μl of 10 × reverse transcription buffer, 0.19 μl of inhibitor (20 U / μl) and 4.16 μl of distilled water were mixed and reacted at 16 ° C for 30 minutes using a DNA amplifier, followed by reaction at 42 ° C for 30 minutes, followed by reaction at 85 ° C for 5 minutes Lt; 0 > C.

(b) Real-time PCR clamping with cDNA synthesized in (a) to select optimal concentration and PNA probe for comparison of specific miRNA expression. 1 μl of a TaqMan MicroRNA assay (ABI, Canada) containing clamping sense primer, antisense primer and TaqMan probe, 1 μl of 1 clamping probe (100 μM) of the probes shown in Table 1, 1 μl of TaqMan 2X universal 10 μl of PCR Master Mix (ABI, Canada), 6.67 μl of distilled water and the like were mixed and mixed at 95 ° C. using a real-time PCR machine (CFX96 ™ Real-Time PCR System, Bio-RAD, USA) The reaction was carried out for 10 minutes, followed by denaturation at 95 ° C for 15 seconds, and binding and elongation at 60 ° C for 60 seconds. Fluorescence was measured at the 60 < 0 > C binding and elongation reaction step. The results for each probe are shown in FIG.

[ Example  4] miRNA  For subtyping miRNA  For the distinction of subtypes PNA The probe  Real time PCR Clamping  Establish method ( RNA clamping ; 2)

 (a) cDNA was prepared to perform real-time PCR clamping with the synthesized miRNA. 1 μl of 1 clamping probe (100 μM) of the probes shown in 25-27 of Table 1, 3 μl of cDNA synthesis primer, 5 μl of dNTP mix (0 μl, 0.8 μl, (100 μM), 1 μl of reverse transcriptase, 1.5 μl of 10 × reverse transcription buffer, 0.19 μl of RNase inhibitor (20 U / μl) and 3.16 μl of distilled water were mixed and reacted at 16 ° C. for 30 minutes using a DNA amplifier Followed by reaction at 42 ° C for 30 minutes, followed by reaction at 85 ° C for 5 minutes, and then allowed to stand at 4 ° C.

(b) Real-time PCR clamping with cDNA synthesized in (a) to find optimal concentration and PNA probe for comparison of specific miRNA expression. 1 μl of a TaqMan MicroRNA assay (ABI, Canada), 10 μl of TaqMan 2X universal PCR master mix (ABI, Canada) and 7.67 μl of distilled water 7.67 μl containing synthetic miRNA as a starting material, The reaction mixture was reacted at 95 ° C. for 10 minutes using a real-time PCR machine (CFX96 ™ Real-Time PCR System, Bio-RAD, USA) The binding and elongation were repeated 40 times at 60 < 0 > C for 60 seconds. Fluorescence was measured at the 60 < 0 > C binding and elongation reaction step.

[ Comparative Example  One] hsa - miR -10a, hsa - miR -10b, hsa - miR -18a, hsa - miR -196a, hsa Comparison with prior art for miR-196b detection-1

The miRNA expression detection method reported in the literature (Nucleic Acids Res. 2005, 33 (20): e179) and the real-time PCR clamping method using the PNA probe according to the present invention were compared. First, by using a mixture solution containing 0 pg, 100 pg, 200 pg and 300 pg of subtype hsa-miR-18b at 300 pg, 200 pg, 100 pg and 0 pg of hsa-miR-18a hsa-miR-18a expression was detected, and the present method was applied to the detection of hsa-miR-18a expression using the same mixed solution. Expression detection for hsa-miR-10a, hsa-miR-10b, hsa-miR-196a and hsa-miR-196b was performed in this manner. The results show ΔC _t between 0 pg and 300 pg in FIG.

As shown in FIG. 2, hsa-miR-18a, hsa-miR-196a and hsa-miR-196b showed no significant difference between the conventional method and the present invention, Shows that the ΔC _t value of the present invention is significantly different from that of the conventional method. These results show that the present invention can detect expression very sensitively.

[ Comparative Example  2] has - miR -10a and has - miR Comparison with prior art for detection of -10b -2

The synthesized miRNA is present at a ratio of 0%, 10%, 20%, 50%, 80%, 90% and 100% of the target hsa-miR-10a, Real time PCR clamping of hsa-miR-10a was performed using the SEQ ID NO: 22 PNA clamping probe in combination. Real-time PCR clamping of has-miR-10b was also performed using the same mixed synthetic miRNA and using the PNA clamping probe of SEQ ID NO: The correlation between Ct values according to each target concentration was analyzed and the accuracy was confirmed.

The results of using SEQ ID NO: 22 are shown in FIG. 4, and the results of using SEQ ID NO: 17 are shown in FIG.

As can be seen from FIG. 4, the higher the relative hsa-miR-10a ratio in solution, the more the difference from the Ct value of 0% was constantly increased, and the difference between the amount of hsa-miR-10a and the Ct value And it is confirmed that there is a correlation. In addition, the above results show that the high ΔC _t Value was found to be due to the low nonspecificity of detection in the 100% sample of the subtype hsa-miR-10b. This is also the result of confirming that the PNA clamping probe of the present invention minimizes nonspecific reaction in detecting miRNA expression and enables more accurate expression comparison.

FIG. 5 shows the result of comparing the present invention with the conventional method by performing real-time PCR clamping for hsa-miR-10b. As can be seen from the results of FIG. 5, the difference ΔC _t between the conventional method and the present invention It is confirmed that the non-specific detection by the existing method is very large, and it can be interpreted that the non-specific detection is minimized by the present invention.

[ Comparative Example  3] has - miR -18a and has - miR Comparison with prior art for detection of -18b -3

The synthesized miRNA is present at a ratio of 0%, 10%, 20%, 50%, 80%, 90% and 100% of the target hsa-miR-18a and the remaining ratio includes the synthesized miRNA Target hsa-miR-18b Real time PCR clamping of hsa-miR-18a was performed using the SEQ ID NO: 34 PNA clamping probe in combination. Real-time PCR clamping of has-miR-18b was also performed using the same mixed synthetic miRNA and using SEQ ID NO: 27 PNA clamping probe. The correlation between the Ct values according to the target hsa-miR-18a concentration was analyzed and the accuracy was confirmed. The results are shown in Figs.

As can be seen in FIG. 6, there is a correlation between the amount of hsa-miR-18a and the Ct value, as the relative hsa-miR-18a ratio in the solution increases constantly from the Ct value of 0% Respectively. In addition, compared with the conventional method, very high ΔC _t The reason for this is that the nonspecific detection is very low in a sample in which 100% of the subtype hsa-miR-18b is present. Therefore, the PNA clamping probe of the present invention minimizes nonspecific reaction in detecting miRNA expression, It is possible.

FIG. 7 shows the result of comparing the present invention with the conventional method by performing real-time PCR clamping for hsa-miR-18b. As can be seen in Figure 7, the C △ _t between the present invention and the conventional method And it is confirmed that the non-specific detection by the existing method is very large, and it can be interpreted that the non-specific detection is minimized by the present invention.

[ Comparative Example  4] has - miR Comparison with prior art for detection of -181a -4

The synthesized miRNA Target hsa-miR-181a was present at a ratio of 0%, 10%, 20%, 50%, 80%, 90% and 100% Real-time PCR clamping of hsa-miR-181a was performed using a PNA clamping probe such as SEQ ID NOS: 44, 50, 56, etc. in a sample mixed with miR-181c and hsa-miR-181d. The correlation between the Ct values according to the target hsa-miR-181a concentration was analyzed and the accuracy was confirmed. The results are shown in Fig.

As can be seen in FIG. 8, there is a correlation between the amount of hsa-miR-181a and the Ct value, as the relative ratio of hsa-miR-181a in solution increases to the value of Ct in 0% . In addition, in the above results, it was found that a very high ΔC _t The results show that miRNAs such as hsa-miR-181b, hsa-miR-181c, and hsa-miR-181d, which are subtypes, are very low in nonspecific detection in 100% samples. This is the result of confirming that the PNA clamping probe of the present invention minimizes nonspecific reaction in detection of miRNA expression and enables more accurate expression comparison.

<110> Panagene Inc. <120> Methods and kits for expression profiling of microRNA using PNA          mediated Real-Time PCR clamping <160> 64 <170> Kopatentin 1.71 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CmiR-let7-a-1 <400> 1 gtagtaggtt gtgtggtt 18 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CmiR-let7-a-2 <400> 2 gtaggttgtg tggtt 15 <210> 3 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> CmiR-let7-a-3 <400> 3 ggttgtgtgg tt 12 <210> 4 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> CmiR-let7-a-4 <400> 4 taggttgtgt 10 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CmiR-let7-c-1 <400> 5 gtagtaggtt gtatggtt 18 <210> 6 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CmiR-let7-c-2 <400> 6 gtaggttgta tggtt 15 <210> 7 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> CmiR-let7-c-3 <400> 7 ggttgtatgg tt 12 <210> 8 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> CmiR-let7-c-4 <400> 8 taggttgtat g 11 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CmiR-let7-g-1 <400> 9 gtagtagttt gtacagtt 18 <210> 10 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CmiR-let7-g-2 <400> 10 gtagtttgta cagtt 15 <210> 11 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> CmiR-let7-g-3 <400> 11 gtttgtacagt tt 12 <210> 12 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> CmiR-let7-g-4 <400> 12 tagtttgtac a 11 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CmiR-10a-1 <400> 13 cctgtagatc cgaatttg 18 <210> 14 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CmiR-10a-2 <400> 14 tgtagatccg aattt 15 <210> 15 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> CmiR-10a-3 <400> 15 gtagatccga a 11 <210> 16 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> CmiR-10a-4 <400> 16 ctgtagatcc ga 12 <210> 17 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CmiR-10a-5 <400> 17 aattcggatc tacag 15 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CmiR-10b-1 <400> 18 cctgtagaac cgaatttg 18 <210> 19 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CmiR-10b-2 <400> 19 tgtagaaccg aattt 15 <210> 20 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> CmiR-10b-3 <400> 20 gtagaaccga a 11 <210> 21 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> CmiR-10b-4 <400> 21 ctgtagaacc ga 12 <210> 22 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CmiR-10b-5 <400> 22 aattcggttc tacag 15 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CmiR-18a-1 <400> 23 gtgcatctag tgcagatag 19 <210> 24 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> CmiR-18a-2 <400> 24 tagtgcagat ag 12 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CmiR-18a-3 <400> 25 ggtgcatcta gtgcagatag 20 <210> 26 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> CmiR-18a-4 <400> 26 gtgcagatag 10 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CmiR-18a-5 <400> 27 tatctgcact agatgcac 18 <210> 28 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> CmiR-18a-6 <400> 28 tatctgcact atatgc 16 <210> 29 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CmiR-18a-7 <400> 29 tatctgcact atatg 15 <210> 30 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CmiR-18b-1 <400> 30 gtgcatctag tgcagttag 19 <210> 31 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> CmiR-18b-2 <400> 31 tagtgcagtt ag 12 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CmiR-18b-3 <400> 32 ggtgcatcta gtgcagttag 20 <210> 33 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> CmiR-18b-4 <400> 33 gtgcagttag 10 <210> 34 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> CmiR-18b-5 <400> 34 ctaactgcac tagatgc 17 <210> 35 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181a-1 <400> 35 actcaccgac agcgt 15 <210> 36 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181a-2 <400> 36 tcaccgacag cgt 13 <210> 37 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181a-3 <400> 37 tcaccgacag cgttgaat 18 <210> 38 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181a-4 <400> 38 accgacagcg ttgaatgt 18 <210> 39 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181a-5 <400> 39 acagcgttga at 12 <210> 40 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181b-1 <400> 40 cccaccgaca gcaat 15 <210> 41 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181b-2 <400> 41 ccaccgacag caat 14 <210> 42 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181b-3 <400> 42 ccaccgacag caatgaat 18 <210> 43 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181b-4 <400> 43 caccgacagc aatgaat 17 <210> 44 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181b-5 <400> 44 ccgacagcaa tgaat 15 <210> 45 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181b-6 <400> 45 cccaccgaca gcaa 14 <210> 46 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181c-1 <400> 46 actcaccgac aggtt 15 <210> 47 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181c-2 <400> 47 actcaccgac aggt 14 <210> 48 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181c-3 <400> 48 tcaccgacagta gttgaatg 18 <210> 49 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181c-4 <400> 49 accgacaggt tgaatgtt 18 <210> 50 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181c-5 <400> 50 ccgacaggtt gaat 14 <210> 51 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181c-6 <400> 51 actcaccgac agg 13 <210> 52 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181d-1 <400> 52 aacccaccga caaca 15 <210> 53 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181d-2 <400> 53 aacccaccga caac 14 <210> 54 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181d-3 <400> 54 ccaccgacaa caatgaat 18 <210> 55 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181d-4 <400> 55 caccgacaac aatgaat 17 <210> 56 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181d-5 <400> 56 ccgacaacaa tgaatgt 17 <210> 57 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> CmiR-181d-6 <400> 57 ccaccgacaa caat 14 <210> 58 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> CmiR-196a-1 <400> 58 tagtttcatg tt 12 <210> 59 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CmiR-196a-2 <400> 59 gtagtttcat gttgttgg 18 <210> 60 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CmiR-196a-3 <400> 60 taggtagttt catgttgt 18 <210> 61 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> CmiR-196a-4 <400> 61 gtttcatgtt g 11 <210> 62 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> CmiR-196b-1 <400> 62 tagtttcctg tt 12 <210> 63 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CmiR-196b-2 <400> 63 gtagtttcct gttgttgg 18 <210> 64 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CmiR-196b-3 <400> 64 taggtagttt cctgttgt 18

Claims (9)

(1) synthesizing cDNA from total RNA or target microRNA (microRNA, miRNA) to be detected;
(2) a PNA (Peptide) which can bind the cDNA perfectly to a clamping primer set of a specific miRNA and a single nucleotide differentiation of the miRNA, and specifically binds to a region containing a different base sequence among subtypes of the miRNA Performing real-time polymerase chain reaction (PCR) on the miRNA in the presence of a Nucleic Acid clamping probe; And
(3) analyzing the result of the real-time PCR to confirm miRNA expression:
Wherein the PNA clamping probe is any one selected from SEQ ID NOs: 23 to 34 designed to bind to the has-miR-18a subtype or has-miR-18b subtype completely. The method according to claim 1,
Wherein the miRNA expression pattern is analyzed by measuring C _t (cycle threshold) value of real-time PCR and comparing miRNA expression or miRNA expression level. delete 3. The method according to claim 1 or 2,
Wherein the cDNA of step (1) is synthesized in the presence of a universal primer or a reverse primer which is a specific miRNA-specific primer. 3. The method according to claim 1 or 2,
The cDNA of step (1) can bind to the reverse primer and the subtype of the miRNA, and the presence of a PNA (Peptide Nucleic Acid) clamping probe that specifically binds to a site containing a different base sequence among the subtypes of the miRNA Lt; RTI ID = 0.0 &gt; miRNA &lt; / RTI &gt; subtype. delete delete 3. The method according to claim 1 or 2,
The miRNA expression pattern is analyzed by comparing the gene amplification with one or more methods selected from the TaqMan probe method, the intercalating method and the molecular beacon (Molecular Beacon) method. Way. delete

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