KR20180059046A - A Method for Enhancing Extraction Efficiency of miRNA in Blood by the Addition of Glycogen and tRNA from yeast - Google Patents

Abstract

The present invention relates to a method for increasing efficiency in extraction of miRNA present in a trace amount in plasma by addition of glycogen and yeast-derived tRNA. According to the present invention, it is possible to quantitatively analyze miRNAs present in a trace amount in a sample in a short time. In addition, since the miRNA detection method by adding glycogen and yeast-derived tRNA is at least three times higher than a case involving addition of glycogen alone or yeast-derived tRNA alone, the method can be efficiently used for detecting various miRNAs.

Description

A method for enhancing the extraction efficiency of miRNA in blood by adding glycogen and yeast-derived tRNA,

The present invention relates to a method for detecting miRNA present in a very small amount in plasma samples and a kit for detecting the miRNA. More specifically, when extracting miRNA by using glycogen and yeast-derived tRNA as carrier as the carrier and optimizing the incubation conditions, it is possible to maximize extraction efficiency of miRNA, And can be used effectively to identify the mechanism of the disease.

MicroRNAs (miRNAs) are very small, unencrypted RNAs, usually composed of 21 to 22 nucleotides, consisting of a single sequence. They regulate other genes by interfering with the translation of mRNA, Embryo development, metabolic regulation and cancer development. Approximately 30% of the total gene is presumed to be regulated by miRNAs. miRNAs are generated through the transcription of individual genes in the unencrypted portion. miRNAs are transcribed from pri-miRNA, a precursor that is transcribed by RNA polymerase II in the nucleus. It is then digested by a nucleic acid-degrading enzyme III called Drosha (dsRNA-specific ribonuclease) to form a hairpin-like pre-miRNA. The hairpin of pre-miRNA is transported out of the cell nucleus by the protein exontin-5 and Ran-GTP as cofactors, and by the action of ribonuclease Ⅲ Dicer and transactivation-responsive RNA binding protein (TRBP) And treated with miRNA duplexes consisting of approximately 22 nucleotides. miRNA duplex binds to RICS (RNA induced silencing complexes) to regulate genes by degrading mRNA or blocking the detoxification process.

Many types of miRNAs and their regulated genes can predict the important role of miRNAs in the mechanisms of various diseases. Therefore, miRNAs are recognized as biomarkers that can be used for diagnosis, prediction and prognosis of diseases by showing the increase or decrease of abnormal miRNA expression according to various diseases such as cancer, diabetes and cardiovascular disease. In addition, the development of miRNA-based therapies has increased significantly.

MiRNAs are present in very small amounts in biological materials and appropriate extraction methods as well as selective and sensitive methods are needed to detect them. Reverse transcription polymerase chain reaction (RT - PCR) is widely used as a detection method of miRNA. The reverse transcription polymerase chain reaction (PCR) is a very good quantitative assay, but it is expensive and involves amplification to analyze very small amounts of miRNAs, which leads to reproducibility and analysis time of 3-4 hours have. Therefore, other assays other than reverse transcription polymerase chain reaction (PCR) are being investigated. Among them, capillary electrophoresis is an excellent method for quantitative, reproducible and separable analysis and most of all it has a short analysis time of less than 1 hour. .

However, these methods have a very low concentration of miRNA in blood, which makes it difficult to analyze accurately and there is a limitation that miRNA concentration variation is large depending on the analysis method or extraction method. Therefore, it is required to develop an extraction method that can maximize miRNA extraction efficiency so that the amount of miRNA present in the blood can be accurately measured.

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.

1. United States Patent Application No. 2012-0034612 (2012.02.09)

1. Niu Y, Zhang L, Qiu H, et al, Scientific report, 2015 5: 15100.

The present inventors have sought to establish a method for detecting miRNAs present in trace amounts in plasma with high sensitivity within a short time. As a result, by adding glycocon and yeast-derived tRNA as miRNA carriers and optimizing their concentration and incubation conditions, it is possible to maximize the precipitation efficiency of RNA including plasma miRNA, thereby detecting miRNAs present in the blood with high sensitivity The present invention has been completed.

Accordingly, it is an object of the present invention to provide a method for increasing the extraction efficiency of miRNA present in blood in a very small amount by adding glycogen and yeast-derived tRNA.

Another object of the present invention is to provide a kit for detecting 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 method for increasing the extraction efficiency of miRNA in blood by adding glycogen and yeast-derived tRNA comprising the steps of:

(a) separating the supernatant after adding the trisol reagent in a plasma sample and centrifuging it;

(b) adding glycocon and yeast-derived tRNA to the supernatant as a carrier and incubating to precipitate miRNA in the blood;

(c) centrifuging to obtain precipitated miRNA pellet extract; And

(d) detecting the miRNA from the miRNA pellet extract.

The present inventors have made efforts to establish a method for detecting miRNAs present in trace amounts in plasma with high sensitivity within a short period of time. As a result, when the glycocon and yeast-derived tRNAs are used together as a carrier for precipitation of miRNA, Of miRNAs can be detected with high sensitivity.

miRNA (microRNA) can play an important role in predicting various diseases. In other words, miRNA can be used as a biomarker that can be used to predict the diagnosis and prognosis of diseases by confirming the increase or decrease of abnormal miRNA expression in various diseases such as cancer, diabetes and cardiovascular disease. However, there are very few miRNAs in the body, and selective and sensitive methods are needed to detect them. Microarrays and reverse transcription polymerase chain reaction (PCR) are widely used as detection methods for miRNAs, but microarrays are capable of detecting many kinds of miRNAs, but they have a disadvantage in that they have poor quantitative properties and reverse transcription polymerase chain In the case of the reaction test, the cost is high and the reproducibility is problematic, and the analysis time is longer than 3-4 hours. In addition, although the method using the triazole (Trizol) is used as an RNA extraction method, the amount of miRNA extracted is limited due to the very small amount of miRNA.

In the present invention, glycogen and yeast-derived tRNA are added as a carrier to improve the precipitation efficiency of miRNA in blood.

According to the detection method developed by the present inventors, it is possible to detect miRNAs present in plasma in a very small amount in a short time.

FIG. 1 shows a process for extracting miRNA in plasma to quantitatively or qualitatively analyze miRNA in plasma. Hereinafter, the present invention will be described step by step.

Step (a): In the plasma sample Trisol  After adding the reagent and centrifuging The supernatant  detach

The first step is to separate the supernatant after adding centrifugation reagent and chloroform in the plasma sample and centrifuging. Trizol reagent is a widely known reagent commonly used in RNA / DNA / protein extraction, and is composed of phenol, guanidinium thiocyanate, and the like. The amount of the triazol reagent to be added in the present invention may vary depending on the purpose, and it may preferably include 0.01 to 100 ml, more preferably 0.1 to 10 ml.

Step (b): Glycocene (Glycogen) and yeast derived tRNA  Addition and / Incubation

In this step, one of the greatest features of the present invention is to maximize the precipitation efficiency of miRNA in blood when the miRNA extraction method using only trizol is added with glycogen and yeast-derived tRNA as a carrier and incubation, It improves the extraction of miRNA in the blood. More specifically, glycogen and yeast-derived tRNAs are used to increase the recovery of nucleic acids or oligonucleotides from alcohol precipitation. In particular, glycogen and yeast-derived tRNAs do not dissolve in ethanol or isopropanol and trap the nucleic acid to form a precipitate. After centrifugation, the unreacted glycogen and yeast-derived tRNA / nucleic acid precipitate form visible pellets, allowing the nucleic acid to be recovered.

In the present invention, miRNA is extracted by further adding isopropanol after RNA layer separation using Glycogen and yeast-derived tRNA, and adjusting incubation time and temperature to aid precipitation formation.

In addition, in the present invention, glycogen and yeast-derived tRNA are preferably used at the same time. The simultaneous use of glycogen and yeast-derived tRNAs results in an increase in extraction efficiency of at least three times as compared to the case of using glycogen alone or yeast-derived tRNA alone (see FIGS. 5A and 5B).

The amount of glycogen used in the present invention is preferably 10 to 100 ug, more preferably 25 to 100 ug, and most preferably 25 to 50 ug per 250 쨉 l of the plasma sample. If the amount of glycogen used is less than 10 ug or more than 100 ug, there may be no significant difference in the amount of miRNA extracted.

The amount of the yeast-derived tRNA to be used is preferably 300 to 1200 ng, more preferably 450 to 1200 ng, and most preferably 600 to 1200 ng for 250 μl of the plasma sample. If the amount of yeast-derived tRNA used is less than 300 ug or exceeds 1200 ug, there may be no significant difference in the amount of miRNA extracted.

In addition, in order to increase the amount of miRNA extracted, it is preferable to perform incubation after addition of the carrier Glycogen and yeast-derived tRNA. The incubation temperature is preferably -70 to -100 ° C, more preferably -70 At a temperature of -80 占 폚, the incubation time is preferably 30 minutes to 3 hours, more preferably 1 hour to 3 hours, and most preferably 1 hour. If the incubation time is less than 30 minutes or more than 3 hours, there may be no significant difference in miRNA extraction.

Step (c): Precipitating by centrifugation miRNA  Obtain pellet extract

When the incubation is completed, the miRNA-containing pellet extract is obtained by centrifugation. A high speed or low speed centrifuge may be used to obtain the pellet extract. Those skilled in the art will be able to select suitable centrifuges and centrifugation times to suit the intended purpose and, if using a Gyrozen centrifuge, with a centrifuge at 1,000 to 30,000 RCF, preferably 10,000 to 15,000 RCF, for 5 to 30 minutes, Preferably 10 minutes to 20 minutes.

Step (d): miRNA  From pellet extract miRNA  detection

Finally, miRNA is detected from the miRNA pellet extract.

The efficient miRNA detection method in which the glycogen and yeast-derived tRNA carriers of the present invention are added is characterized in that the amount of the miRNA extracted is at least 3 (as compared with the case of adding glycogen alone or yeast-derived tRNA alone) Fold, it can be easily and efficiently detected using a variety of miRNA detection methods known in the art. On the other hand, when glycogen and yeast-derived tRNA are not added, miRNA pellet extract is sometimes present and sometimes there is a problem in reproducibility. Therefore, there is a limit to carry out the experiment without adding them. Therefore, it was found that the present invention can improve not only the reproducibility but also the detection efficiency of miRNA when using glycogen and yeast-derived tRNA at the same time.

According to a preferred embodiment of the present invention, step (d) comprises the steps of: (d-1) hybridizing a miRNA pellet extract and a free DNA conjugated with a fluorescent substance specific to the miRNA expected to be present in the blood Hybridizing in a buffer; (d-2) isolating and identifying a DNA-miRNA complex using a capillary electrophoresis (CE / LIF) system equipped with a laser induced fluorescence (LIF) detector; And (d-3) confirming the presence of miRNA in blood by confirming the DNA-miRNA complex peak as a result of step (d-2).

The detection method of the present invention can be used to predict the diagnosis and prognosis of various diseases.

The fluorescent label may provide a signal for detecting hybridization, which may be linked to an oligonucleotide. Suitable labels that can be used in the present invention include various fluorescent materials such as fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia) 5'-carboxyfluorescein phosphoramidite (6-FAM) may be used. Markers can be obtained by a variety of methods routinely practiced in the art, such as the nick translation method, the Multiprime DNA labeling systems booklet (Amersham (1989)) and the kaination method (Maxam & Gilbert, Methods in Enzymology , ≪ / RTI > 65: 499 (1986)).

Suitable conditions for hybridization include, but are not limited to, Nucleic Acid Hybridization, A Practical Approach , Hayes, BD, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, IRL Press, Washington, DC (1985). The stringent condition used for hybridization can be determined by controlling the temperature, the ionic strength (buffer concentration) and the presence of a compound such as an organic solvent, and the like. This stringent condition can be determined differently depending on the sequence to be hybridized. Various types of buffers used in the present invention can be used for the buffer used in the present invention, and a buffer having the highest degree of hybridization among the buffers can be selected as an optimal hybridization condition. For example, the hybridization buffer of the present invention may use a TNM buffer, a PBS buffer Tris-Cl buffer, an SSC buffer, a HEN buffer, or a TEN buffer, preferably a TEN buffer.

According to a preferred embodiment of the present invention, the capillary electrophoresis system of the present invention is equipped with a laser induced fluorescence (LIF) detector (CE / LIF system).

In the present invention, when the CE / LIF system is used, the hybridized DNA-miRNA complex can be isolated using an uncoated capillary. The capillary preferably has an inner diameter of 50-100 mu m and a length of 20-60 cm, but is not limited thereto. The hybridized DNA-miRNA complex can be isolated when a voltage of 10-20 kV, preferably 14 kV, is applied in the capillary using a Tris-borate buffer as a separation buffer.

The wavelength of the LIF detector may vary depending on the kind of the fluorescent material to be detected, and when 5'-carboxyl fluorosynthosphoramidite (6-FAM) is used as the fluorescent material, the excitation wavelength is preferably 400-500 nm , Most preferably 488 nm, and the emission wavelength preferably has 500-600 nm, most preferably 520 nm.

According to another preferred embodiment of the present invention, the step (d) may be performed by amplifying miRNA using qRT-PCR to analyze miRNA.

According to another aspect of the present invention, the present invention provides a kit for detecting the miRNA.

Since the kits of the present invention comprise the miRNAs, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

According to a preferred embodiment of the present invention, the kit of the present invention is a kit for detecting miRNA in plasma, comprising: (a) a triazole reagent; (b) glycocene; (c) yeast-derived tRNA; (d) a fluorescent substance capable of hybridizing to the miRNA; (e) a hybridization buffer; And (f) a buffer for DNA-miRNA complex separation.

According to a preferred embodiment of the present invention, the hybridization buffer is one or more buffers selected from the group consisting of TNM buffer, PBS buffer, Tris-Cl buffer, SSC buffer, HEN buffer and TEN buffer. More preferably a TEN buffer.

According to a preferred embodiment of the present invention, the separation buffer is a tris-borate buffer.

According to a preferred embodiment of the present invention, the fluorescent substance is 5'-carboxylfluorosynphosphoramidite (6-FAM).

The features and advantages of the present invention are summarized as follows:

(i) The present invention provides a method for increasing the efficiency of detecting minute miRNA present in plasma by glycogen and yeast-derived tRNA.

(Ii) According to the present invention, it is possible to quantitatively analyze miRNA present in a very small amount in a sample in a short time.

(Iii) Further, the method of detecting miRNA by adding glycogen and yeast-derived tRNA of the present invention can be carried out by using glycogen and yeast-derived tRNA at the same time and optimizing incubation conditions to obtain glycogen and yeast-derived tRNA alone Which is about three times higher than that of wild-type miRNAs.

FIG. 1 shows a process for extracting miRNA in plasma to quantitatively or qualitatively analyze miRNA in plasma.
FIG. 2 (a) is a graph showing the relationship between the size of the DNA-miRNA complex peak (miRNAs) and the size of the DNA-miRNA complex peaks, as a result of detection using glycogen alone and hybridization with a single stranded DNA having a fluorescent residue Was found to increase with the increase in glycogen usage and 25 ug showed the highest sensitivity.
FIG. 2b shows the result of detection of the yeast-derived tRNA by using different amounts of yeast-derived tRNA, followed by hybridization with single-stranded DNA having a fluorescent moiety and using CE-LIF, The size (miRNA extraction efficiency) varies with increasing use of yeast-derived tRNA, confirming the highest sensitivity when 1200 ng is used.
FIG. 3 shows the results of detection using glycogen and yeast-derived tRNA at the same time, using glycogen and yeast-derived tRNAs in different amounts, hybridizing with single-stranded DNA having fluorescent residues and using CE-LIF, -mRNA complex peak (miRNA extraction efficiency) varies with increasing use of glycogen and yeast-derived tRNA, and 25 ng of glycogen and 600 ng of yeast-derived tRNA showed the highest sensitivity.
FIG. 4 is a graph comparing the efficiency of miRNA extraction according to incubation conditions after isolating the RNA layer using 20 g of glycogen and 600 ng of yeast-derived tRNA together with the triazole reagent, adding isopropanol, Min, at 80 ° C for 30 minutes, 1 hour, 3 hours, and over night. The result showed that the sensitivity was highest when left at 80 ° C for 1 hour.
FIG. 5A shows the results of confirming the recovery rate of miRNA according to the case where glycogen alone, yeast-derived tRNA alone, or both of them were used by using CE-LIF at 80 ° C as determined in Example 2. As a result, glycogen 25 ug and yeast-derived tRNA at a rate of at least 3 times higher than that of glycogen alone or yeast-derived tRNA when using 600 ng of yeast-derived tRNA at the same time.
Fig. 5B shows the results of the results of qRT-PCR for the recovery of miRNA from glycogen alone, yeast-derived tRNA alone, and simultaneous use thereof at 80 DEG C for 1 hour as determined in Example 2. As a result, glycogen 25 ug, and yeast-derived tRNA at a concentration of 600 ng at the same time, the extraction efficiency was at least 3 times higher than that of glycogen alone or yeast-derived tRNA alone.

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: in the blood miRNA  Glycogen and yeast origins for increased extraction efficiency tRNA  Establish optimal amount

MiRNA 155 (5'-UUAAUGCUAAUCGUGAUAGGGGU-3 ') was added to 250 μl of blood plasma samples, and Trizol® reagent and glycogen alone and yeast-derived tRNA were simultaneously used as carrier to extract miRNA in plasma .

Specifically, miRNA extraction samples were analyzed using CE-LIF. In the case of CE-LIF, the extracted samples were dissolved in the hybridization solution and 5'-carboxyl fluorosynthosphoramidite (miRNA- 6-FAM), denatured at 95 ° C for 5 minutes, hybridized with TEN buffer (50 mM Tris-Ac (pH 8.0), 10 mM EDTA, 50 mM NaCl) at 40 ° C for 15 minutes And analyzed by CE system with LIF detector. The CE system was a PA 800 plus CE system (Beckman Coulter, Fullerton, CA, USA) and the LIF detector was a source and filter with excitation and emission wavelengths of 488 nm and 520 nm, respectively. Separation was carried out by applying a voltage of 14 kV in an uncoated capillary (Beckman Coulter) with an inner diameter of 75-μm and a length of 50-cm using 125 mM Tris-borate buffer (pH 10.0).

FIG. 2A shows the case where glycogen alone was used, and the sensitivity was changed when the amount of glycogen used was increased. FIG. 2B shows that when the yeast-derived tRNA was used alone, And it was confirmed that the highest sensitivity was obtained when 1200 ng was used.

However, the results of FIG. 3 confirm that the extraction efficiency of glycogen and yeast-derived tRNA is at least 3 times higher than that in the case of using alone in the case of using tRNA at the same time. In particular, 25 ug of glycogen and 600 ng of yeast-derived tRNA showed the highest sensitivity.

Example  2: Serum miRNA  Optimal for effective extraction Incubation  Time and temperature Sho Lip

After 250 μl of plasma sample, miRNA 155 was added, and the RNA-containing solution was separated using Trizol® reagent, glycogen and yeast-derived tRNA, added with isopropanol, and incubated at different temperatures and times miRNA was extracted. Extracted samples were analyzed using CE-LIF and performed under the same conditions as in Example 1.

FIG. 4 shows that the extraction efficiency of miRNA varies depending on the incubation conditions, and when it is left at 80 ° C for about 1 hour, it is confirmed that the DNA peak associated with the miRNA is the highest.

Example  3: in plasma miRNA  The extraction recovery rate was measured by CE- LIF  Confirm using

After addition of miRNA-155 to plasma samples (250 μl), Trizol® reagent, glycogen alone, and yeast-derived tRNA, which are used for the extraction of RNA or miRNA, After adding isopropanol, incubation was carried out at 80 ° C for 1 hour, and total RNA including miRNA was extracted in the form of a pellet. In order to analyze using CE-LIF, the extracted total RNA was hybridized with a DNA probe containing 6-FAM, which is a fluorescent substance specific to miRNA-155, and then ligated, / LIF.

FIG. 5A is a graph showing the recovery rate of miRNA according to the case of using glycogen alone, yeast-derived tRNA alone, and CE-LIF when using them simultaneously. When 25 g of glycogen and 600 ng of yeast-derived tRNA are used simultaneously, glycogen alone, the extraction efficiency was increased by at least 3 times as compared with the case of using tRNA alone.

Example  4: in plasma miRNA  The extraction recovery rate qRT - PCR  Confirm using

After addition of miRNA-155 to plasma samples (250 μl), Trizol® reagent, glycogen alone, and yeast-derived tRNA, which are used for RNA or miRNA extraction, After adding isopropanol, incubation was carried out at 80 ° C for 1 hour, and total RNA including miRNA was extracted in the form of a pellet. For analysis using qRT-PCR, total RNA samples were synthesized using Mir-X miRNA First-Strand Synthesis Kit (Takara, Dalian, China). The synthesized cDNA was amplified by quantitative real-time PCR (Bio-Rad, USA) using a SYBR Green Real-time PCR Master Mix and a forward primer (5'-TTAATGCTAATCGUGATAGGGGT-3 ') of miRNA 155 and a universal reverse primer Expression of miRNA 155 was analyzed.

FIG. 5b shows the results of qRT-PCR for the recovery of miRNAs from glycogen alone, yeast-derived tRNA alone, and simultaneous use thereof. When 25 g of glycogen and 600 ng of yeast-derived tRNA were used simultaneously, glycogen alone, yeast- it was confirmed that the extraction efficiency was at least 3 times higher than that in the case of using tRNA alone.

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.

<110> Korea Institute of Science & Technology <120> A Method for Enhancing Extraction Efficiency of miRNA in Blood by          Addition of Glycogen and tRNA from yeast <130> DP-2016-0688 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> miRNA155 <400> 1 uuaaugcuaa ucgugauagg ggu 23

Claims (12)

A method for increasing the extraction efficiency of existing miRNA in the blood by adding glycogen and yeast-derived tRNA comprising the steps of:
(a) separating the supernatant after adding the trisol reagent in a plasma sample and centrifuging it;
(b) adding glycogen and yeast-derived tRNA to the supernatant as a carrier and incubating to precipitate miRNA in blood;
(c) centrifuging to obtain precipitated miRNA pellet extract; And
(d) detecting the miRNA from the miRNA pellet extract.
The method according to claim 1, wherein the concentration of the glycogen is from 10 to 100 ug in relation to 250 쨉 l of the plasma sample, and from 300 to 1200 ng of the yeast-derived tRNA.
2. The method of claim 1, wherein the incubation is from -60 to -100 DEG C for 30 minutes to 3 hours.
The method of claim 1, wherein step (d) comprises the following steps:
(d-1) hybridizing a miRNA pellet extract and a free DNA conjugated with a fluorescent substance specific to an miRNA expected to be present in blood in a hybridization buffer;
(d-2) isolating and identifying a DNA-miRNA complex using a capillary electrophoresis (CE / LIF) system equipped with a laser induced fluorescence (LIF) detector; And
(d-3) confirming the presence of miRNA in the blood by confirming the DNA-miRNA complex peak as a result of the step (d-2).
5. The method of claim 4, wherein the LIF detector has an excitation wavelength of 400-500 nm and an emission wavelength of 500-600 nm.
5. The method of claim 4, wherein the DNA-miRNA complex is isolated using an uncoated capillary.
7. The method of claim 6, wherein the uncoated capillary has an inner diameter of 50-100 [mu] m and a length of 20-60 cm.
5. The method of claim 4, wherein the separation is performed by applying a voltage of 10-20 kV in an uncoated capillary using a tris-borate buffer.
A kit for detecting miRNA in plasma, comprising: (a) a triazol reagent; (b) glycocene; (c) yeast-derived tRNA; (d) a fluorescent substance capable of hybridizing to the miRNA; (e) a hybridization buffer; And (f) a miRNA detection kit for use in a capillary electrophoresis (CE / LIF) system comprising a buffer for DNA-miRNA complex separation.
10. The kit according to claim 9, wherein the hybridization buffer is one or more buffers selected from the group consisting of TNM buffer, PBS buffer, Tris-Cl buffer, SSC buffer, HEN buffer and TEN buffer.
10. The kit according to claim 9, wherein the separation buffer is a tris-borate buffer.
[Claim 11] The kit for detecting miRNA according to claim 9, wherein the fluorescent substance is 5'-carboxyl fluorosynthosphoramidite (6-FAM).

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