CN103320519A - PCR analysis method for quantitative detection of microRNA - Google Patents

Abstract

The invention discloses a PCR analysis method for the quantitative detection of microRNA based on a base stacking hybridization principle. The method comprises the following steps: stabilizing the combination of a forward primer with a DNA amplification template through a base stacking hybridization effect after the combination of target microRNA with the DNA amplification template, extending the forward primer under the action of a DNA polymerase, initiating a PCR reaction under the combined action of the extended forward primer and a reverse primer to obtain double-stranded DNA, combining a dye SYBR Green I with the double-stranded DNA, determining the fluorescence signal intensity in a reaction system in real time, comparing with a standard work curve, and calculating to obtain the concentration of the target microRNA. The method has the characteristics of high sensitivity, strong specificity, simple operation, low cost and the like, and can be widely used for detecting microRNA in the biological samples of tissues, blood or cells.

Description

PCR analysis method for quantitative detection of microRNA

technical field

The invention belongs to the field of detection and analysis, in particular to a PCR analysis method for quantitative detection of microRNA based on the principle of base stacking hybridization.

Background technique

MicroRNAs are a class of endogenous non-coding RNAs with regulatory functions found in eukaryotes, with a size of about 18-25 nucleotides. Mature microRNAs are produced by a series of nuclease cleavage and processing of longer primary transcripts, and then assembled into RNA-induced silencing complexes, which recognize target mRNAs by base pairing, and according to the degree of complementarity Differently direct the silencing complex to degrade target mRNAs or prevent translation of target mRNAs. microRNAs are involved in a wide variety of regulatory pathways, including development, viral defense, hematopoietic processes, organ formation, cell proliferation and apoptosis, fat metabolism, etc. The abnormal expression of microRNAs is usually closely related to the occurrence and development of cancer and the clinical response to therapeutic drugs, and has become an ideal tumor marker. Therefore, the quantitative analysis of microRNAs is of great significance for understanding the biological functions of microRNAs in cancer development, early diagnosis of cancer, monitoring of curative effect and judgment of prognosis. Compared with traditional nucleic acid detection, the unique characteristics of microRNAs, such as short sequence, high family sequence homology, and low abundance expression, increase the difficulty of its detection. At present, the methods for detecting microRNAs mainly include Northern blot analysis, microarray chip, polymerase chain reaction (PCR), in situ hybridization and so on. From the discovery of microRNAs to the current analysis of microRNAs, Northern blot technology has been applied to the identification of microRNAs and the discovery of new microRNAs. Although Northern blot is the current standard method for microRNA analysis, its disadvantages are cumbersome and time-consuming operation, low sensitivity, a large number of samples and separation and enrichment steps are required for analysis and detection, and it is very sensitive to contamination. Improper operation in every step of the experiment will affect Analyze the results. Although microarray chip technology can achieve high throughput and simultaneous detection of multiple components, its production and detection costs are high; the sample volume available on the chip is small, resulting in low sensitivity; in addition, microRNAs have short sequences and high sequence similarity , the hybridization environment of all microRNAs to be tested cannot be optimized at the same time, so the selectivity is not high. Reverse transcription-polymerase chain reaction (RT-PCR) is the main method for high-sensitivity quantitative detection of microRNAs. Due to the short sequence of microRNAs, it is difficult to design amplification primers, so the analysis of short-sequence microRNAs needs to improve the traditional RT-PCR method. In recent years, a variety of new methods for detecting microRNAs based on RT-PCR signal amplification have been developed, such as primer extension RT-PCR, stem-loop primer RT-PCR, and microRNAs tailing RT-PCR. Although the microRNAs detection method based on RT-PCR reaction has the characteristics of rapidity, strong specificity, and high sensitivity, it requires reverse transcription operation, which undoubtedly increases the cost of the experiment and the complexity of the design. Therefore, it is still a challenge to develop microRNAs analysis technology that is simple and direct, has high sensitivity, strong specificity, wide application range, low detection cost, and accurate and reliable results.

Contents of the invention

Aiming at the deficiencies of the prior art, the object of the present invention is to provide a PCR analysis method for quantitative detection of microRNA based on the principle of base stacking hybridization.

In order to achieve the above object, the technical scheme of the present invention is as follows:

The invention provides a PCR analysis method for quantitative detection of microRNA, comprising the following steps:

After the target microRNA is combined with the DNA amplification template, the combination of the forward primer and the DNA amplification template is stabilized by base stacking hybridization, and the forward primer is extended under the action of DNA polymerase. Under the joint action of the reverse primer, Initiate the PCR reaction to obtain double-stranded DNA. The dye SYBR Green I combines with the double-stranded DNA to generate a fluorescent signal. The intensity of the fluorescent signal in the reaction system is measured in real time. Compared with the standard working curve, the concentration of the target microRNA is calculated.

The target microRNAs are let-7a, miR-141, miR-21, miR-200b.

The let-7a is 5'-UGAGGUAGUAGGUUGUAUAUAGUU-3'; the DNA amplification template sequence is 5'-GGCTAAGACAGATGCTCTTTGCCAACAGGCCACAGAATTCCTACACTCAAAGTCGTACTGAACTATACAACCTACTACCTCATCGCACT-3'.

The miR-141 is 5'-UAACACUGUCUGGUAAAGAUGG-3'; the DNA amplification template sequence is 5'-GGCTAAGACAGATGCTCTTTGCCAACAGGCCACAGAATTCCTACACTCAAAGTCGTACTGCCATCTTTACCAGACAGTGTTATCGCACT-3'.

The miR-21 is 5'-UAGCUUAUCAGACUGAUGUUGA-3'; the DNA amplification template sequence is 5'-GGCTAAGACAGATGCTCTTTGCCAACAGGCCACAGAATTCCTACACTCAAAGTCGTACTGTCAACATCAGTCTGATAAGCTATCGCACT-3'.

The miR-200b is 5'-UAAUACUGCCUGGUAAUGAUGA-3'; the DNA amplification template sequence is 5'-GGCTAAGACAGATGCTCTTTGCCAACAGGCCACAGAATTCCTACACTCAAAGTCGTACTGTCATCATTACCAGGCAGTATTATCGCACT-3'.

The forward primer sequence is 5'-ATGCGACGATGCGA-3'.

Described DNA polymerase is Taq DNA polymerase.

The sequence of the reverse primer is 5'-GGCTAAGACAGATGCTC-3'.

The PCR reaction system includes 50-500 μM dNTPs, 200-800 nM forward primer, 200-800 nM reverse primer, 5-200 nM DNA amplification template, 0.01-0.08 U/μL DNA polymerase, dye SYBR Green I, 0.1 ～1.0U/μL RNase inhibitor, target microRNA and DEPC water.

The procedure of the PCR reaction is: pre-denaturation at 94°C for 30s; denaturation at 94°C for 5s, annealing and extension at 55-64°C for 30s, and 20-40 cycles.

Compared with the prior art, the present invention has the following advantages and good effects:

1. High sensitivity: The target microRNA and the forward primer are hybridized to the DNA amplification template at the same time, so that the PCR amplification reaction can be effectively carried out, and an exponentially amplified fluorescent signal can be generated, thereby greatly improving the detection sensitivity.

2. Good selectivity: When there is a single base difference between the competing microRNA and the target microRNA, it will directly affect the hybridization stability between the forward primer and the DNA amplification template, thereby affecting the efficiency of PCR amplification.

3. Low background signal: When there is no target microRNA, the combination of the forward primer and the DNA amplification template is unstable during PCR annealing and extension, and effective PCR amplification cannot be performed.

4. High-throughput detection: Using eight tubes or 96-well PCR plates, it can realize the simultaneous production of standard working curves and detection of samples to be tested, reducing detection errors.

5. The operation process is simple and fast: the whole operation process only needs one sample addition process.

6. Low detection cost: This method uses cheap DNA polymerase and fluorescent dye SYBR Green I.

7. No pollution: The whole detection process does not need to use organic solvents or toxic reagents.

8. The method of the present invention has the advantages of simple operation, high sensitivity, strong specificity, wide application range, low cost, etc., and is a convenient and practical analytical technique.

Description of drawings

Figure 1 is a schematic diagram of the principle of the PCR analysis let-7a method for quantitative detection of microRNA.

Fig. 2 is a schematic diagram of the prepared let-7a working curve. (A) Amplification curves of let-7a reaction solutions with different concentrations; (B) Working curves of fluorescence threshold (C _t ) and let-7a concentration logarithm of let-7a reaction solutions with different concentrations.

Figure 3 is a graph of a specificity experiment. (A) Amplification curves of different target microRNA reaction solutions; (B) Histogram of experimental data comparison (ΔC _t =C _{t,blank control} -C _t,microRNA ).

Figure 4 is the amplification curve of let-7a analyzed when the PCR system for quantitative detection of microRNA contains different concentrations of amplified templates.

Figure 5 is the amplification curve of let-7a analyzed when the PCR system for quantitative detection of microRNA contains different concentrations of DNA polymerase.

Figure 6 is the amplification curve of let-7a analyzed when the PCR system for quantitative detection of microRNA contains different concentrations of forward and reverse primers.

Fig. 7 is a schematic diagram of the principle of PCR analysis miR-141 method for quantitative detection of microRNA.

Fig. 8 is a schematic diagram of the principle of PCR analysis miR-21 method for quantitative detection of microRNA.

Fig. 9 is a schematic diagram of the principle of PCR analysis miR-200b method for quantitative detection of microRNA.

Detailed ways

Below in conjunction with embodiment the present invention is described in further detail.

Symbol Description:

In Figure 1, let-7a is 5′-UGAGGUAGUAGGUUGUAUAGUU-3′, the amplification template sequence is 5′-GGCTAAGACAGATGCTCTTTGCCAACAGGCCACAGAATTCCTACACTCAAAGTCGTACTGAACTATACAACCTACTACCTCATCGCACT-3′, the forward primer sequence is (5′-ATGCGACGATGCGA-3′), and the reverse primer sequence is ( 5′-GGCTAAGACAGATGCTC-3′).

In Figure 2, C _t is the fluorescence threshold of each reaction solution, which can be set automatically by the fluorescent quantitative PCR instrument or manually. -lg C _let-7a is the negative logarithm of the let-7a concentration in the let-7a standard solution.

In Figure 3, ΔC _t =C _{t,blank control} -C _t,microRNA , where C _{t,blank control} is the fluorescence threshold of the blank sample solution (without microRNA), and C _t,microRNA is the fluorescence threshold of the solution containing microRNA.

In Figure 7, miR-141 is 5′-UAACACUGUCUGGUAAAGAUGG-3′, the amplified template sequence is 5′-GGCTAAGACAGATGCTCTTTGCCAACAGGCCACAGAATTCCTACACTCAAAAGTCGTACTGCCATCTTTACCAGACAGTGTTATCGCACT-3′ forward primer sequence is (5′-ATGCGACGATGCGA-3′), and reverse primer sequence is (5′-ATGCGACGATGCGA-3′) '-GGCTAAGACAGATGCTC-3').

In Figure 8, miR-21 is 5′-UAGCUUAUCAGACUGAUGUUGA-3′, the amplification template sequence is 5′-GGCTAAGACAGATGCTCTTTGCCAACAGGCCACAGAATTCCTACACTCAAAGTCGTACTGTCCAACATCAGTCTGATAAGCTATCGCACT-3′ forward primer sequence is (5′-ATGCGACGATGCGA-3′), and the reverse primer sequence is (5′ '-GGCTAAGACAGATGCTC-3').

In Figure 9, miR-200b is 5′-UAAUACUGCCUGGUAAUGAUGA-3′, the amplification template sequence is 5′-GGCTAAGACAGATGCTCTTTGCCAACAGGCCACAGAATTCCTACACTCAAAGTCGTACTGTCATCATTACCAGGCAGTATTATCGCACT-3′ forward primer sequence is (5′-ATGCGACGATGCGA-3′), and the reverse primer sequence is (5′ '-GGCTAAGACAGATGCTC-3').

The following examples will help to understand the present invention, but the content of the present invention cannot be limited.

The DNA amplification template contains three functional sequences, which are the hybridization sequence with the target microRNA, the hybridization sequence with the forward primer, and the same sequence as the reverse primer; the position is that the 3′ end of the DNA amplification template contains 4 to 7 The base complementary to the forward primer and the base complementary to the target microRNA sequence, the 5' end of the DNA amplification template contains a sequence identical to the sequence of the reverse primer.

The forward primer consists of two parts of sequence: 4-7 bases hybridize between the 3' end sequence of the primer and the 3' end sequence of the template; the 5' end sequence of the primer is an overhang of 6-15 bases.

Base stacking hybridization, also known as continuous stacking hybridization, is a hybridization between two or more consecutive tandem sequences (DNA or RNA) and a longer complementary single-stranded sequence (DNA or RNA). The method is more stable than the method of single sequence and a longer complementary single-stranded sequence. The PCR analysis method for the quantitative detection of microRNA using the base stacking hybridization principle is as follows. Design a DNA amplification template containing three functional sequences: the hybridization sequence to the target microRNAs, the hybridization sequence to the forward primer, and the same sequence to the reverse primer. The position on the DNA amplification template is: the 3' end of the template contains 4 to 7 bases complementary to the forward primer and 18 to 25 bases complementary to the target sequence, and the 5' end of the template contains a section that is complementary to the reverse primer. to the same sequence as the primer sequence. When there are target microRNAs in the reaction system, the target microRNAs and forward primers hybridize to the DNA amplification template in a closely adjacent series, and the hybridization stability of the forward primer and template is improved due to base stacking. The role of the primer triggers the PCR reaction, and a large amount of double-stranded DNA products are amplified. The double-stranded DNA products can be combined with the fluorescent dye SYBR Green I, and emit corresponding fluorescence under the irradiation of incident light of a certain wavelength. The reaction can be measured in real time. The intensity of the amplified fluorescence signal in the system is used to obtain the fluorescence threshold, and compared with the standard working curve, the concentration of target microRNAs is calculated.

The method of the present invention can be further described as follows:

Due to the small number of hybrid bases between the forward primer and the DNA amplification template, it cannot be stably combined with the DNA amplification template at the temperature of annealing and extension. Therefore, after the target microRNAs are combined with the DNA amplification template, the hybridization stability and hybridization efficiency of the forward primer and the template are improved under the effect of base stacking. Under the action of DNA polymerase, the forward primer is extended to obtain double-stranded DNA, and the double-stranded DNA is dissociated under the denaturation process of PCR, releasing the single-stranded DNA amplification template and a new single-stranded DNA. In the process of annealing and extension, the reverse primer is combined to start a new round of PCR reaction, repeating the cycle of denaturation-annealing-extension, and a large number of double-stranded DNA products are obtained. A large number of dyes SYBR GreenI combine with double-stranded DNA products to generate fluorescent signals, real-time The fluorescence signal intensity in the reaction system is measured, and the fluorescence threshold is obtained, compared with the standard working curve, and the concentration of the target microRNA is calculated.

The PCR reaction mixture contains polymerase, RNase inhibitor and dNTPs; the concentration of the polymerase is 0.0125UμL ^-1 ~ 0.05UμL ^-1 , the concentration of RNase inhibitor is 0.1UμL ^-1 ~ 1.0UμL ^-1 , the concentration of dNTPs solution 80-500 μM; the reaction buffer is 50 mM Tris-HCl, 20 mM potassium chloride, 10 mM ammonium sulfate, 2 mM magnesium sulfate, and the pH is 9.0.

The standard working curve is a standard solution of target microRNA with a known concentration. According to the PCR amplification, the intensity of the amplified fluorescence signal in the reaction system is measured in real time and the fluorescence threshold is obtained, and then according to the concentration of microRNA in the standard solution and the concentration in the system Fluorescence threshold to make a standard working curve.

Example 1

The analysis method of let-7a (5′-UGAGGUAGUAGGUUGUAUAGUU-3′) was detected by one-step real-time quantitative PCR based on the principle of base stacking hybridization, and the detection principle is shown in Figure 1. Using TaqDNA polymerase and double-stranded DNA specific dye SYBR Green I. The functional sequence on the DNA amplification template is the same sequence as the reverse primer (5′-GGCTAAGACAGATGCTC-3′), the target let-7a binding sequence (5′-AACTATACAACCTACTACCTCA-3′) and the forward primer binding sequence (5′ -TCGCCT-3').

The following specific implementation of let-7a detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions or the conditions suggested by the manufacturer. The specific operation steps are as follows: add 2μL reaction buffer solution (50mM Tris-HCl, 20mM potassium chloride, 10mM ammonium sulfate, 2mM magnesium sulfate, pH 9.0) and 2μL DNA amplification template to the eight-tube or 96-well PCR plate well solution (5nM), 2μL forward primer solution (600nM), 2μL reverse primer solution (600nM), 1.6μL dNTPs solution (200μM), 0.2μL Taq DNA polymerase solution (0.025U/μL), 0.4μL RNase inhibitor (0.8U/μL), 0.8 μL SYBR Green I solution (0.4×), 2 μL target let-7a solution, 7 μL DEPC water.

Put the above reaction solution in a fluorescent quantitative PCR instrument, pre-denaturation at 94°C for 30s; denaturation at 94°C for 5s, annealing and extension at 60°C for 30s, a total of 30 cycles, the instrument measures the amplified fluorescence signal of the solution in real time, and gives the fluorescence Threshold (Ct).

Preparation of the working curve: Prepare let-7a standard solutions of known concentrations, the concentrations are 500fM, 5pM, 50pM, 100pM, 500pM, 1nM and 10nM, and blank samples (do not contain 1et-7a, that is, let-7a concentration is 0), follow the above steps to measure the amplification curve of each reaction solution, as shown in Figure 2 (A). Then make a standard working curve according to the negative logarithmic value of the concentration of let-7a standard solution and its fluorescence threshold (Ct), as shown in Figure 2 (B), the negative logarithmic value of the concentration of let-7a standard solution and its fluorescence threshold (Ct ) is linear in the range of 500fM～10nM, and the linear equation is C _t =–6.28–2.77lg C _let-7a .

Accompanying drawing 3 shows the specificity experiment of the method of the present invention, select let-7b, let-7d, let-7e, miR-141, miR-21, miR-200b, as let-7a control sample (detection concentration is 100pM ), and a blank sample (without microRNA, that is, the microRNA concentration is 0), the sequence of let-7b is 5′-UGAGGUAGUAGGUUGUGUGGUU-3′, the sequence of let-7d is 5′-AGAGGUAGUAGGUUGCAUAGUU-3′, and the sequence of let-7e is 5′ -UGAGGUAGGAGGUUGUAUAGU-3', the sequence of miR-141 is 5'-UAACACUGUCUGGUAAAGAUGG-3', the sequence of miR-21 is 5'-UAGCUUAUCAGACUGAUGUUGA-3', and the sequence of miR-200b is 5'-UAAUACUGCCUGGUAAUGAUGA-3'. As shown in Figure 3 (A) is the amplification curve of each target reaction solution, as shown in Figure 3 (B) is the difference between the fluorescence threshold of each target reaction solution and the fluorescence threshold of the blank sample (ΔC _t = C _{t, blank control} - C _{t, microRNA} ), the experimental results show that the method has strong specificity.

The amplification template concentration of the PCR system of the method of the present invention can vary within a wide range. The same concentration of let-7a was detected by using the amplification template with a concentration ranging from 5 to 100 nM. Prepare a let-7a solution with a concentration of 100pM, and a blank sample (without 1et-7a, that is, let-7a concentration is 0), and the amplification template concentrations of the reaction solution are 5nM, 10nM, 50nM and 100nM respectively. Other conditions were operated according to the above steps, and the amplification curves of each reaction solution were measured. As shown in Figure 4, the concentration of the amplified template was in the range of 5-100nM, and the content of 100pM let-7a could be detected. It can be concluded from Figure 4 that when the template concentrations were 5nM, 10nM, 50nM, and 100nM, the ΔC _t values corresponding to the detection of 100pM let-7a were 4.31, 3.42, 3.17, and 2.60, respectively.

The concentration of DNA polymerase in the PCR system of the method of the present invention can vary within a wide range. Let-7a at the same concentration was detected by using DNA polymerase with a concentration ranging from 0.0125 to 0.05 UμL ^-1 . Prepare a let-7a solution with a concentration of 100pM, and a blank sample (without 1et-7a, that is, the let-7a concentration is 0), and the DNA polymerase concentrations in the reaction solution are 0.0125UμL ^-1 , 0.025UμL ^-1 , and 0.0375UμL ^-1 and 0.05 U μL ^-1 . Other conditions were operated according to the above steps, and the amplification curves of each reaction solution were measured. As shown in Figure 5, the concentration of DNA polymerase in the range of 0.0125 ~ 0.05UμL ^-1 can detect the content of 100pM let-7a. It can be concluded from Figure 5 that when the concentration of DNA polymerase is 0.0125UμL ^-1 , 0.025UμL ^-1 , 0.0375UμL ^-1 , and 0.05UμL ^-1 , the ΔC _t values corresponding to the detection of 100pM let-7a are 2.57 and 4.67 respectively , 3.60, 4.49.

The concentration of the forward and reverse primers of the PCR system of the method of the present invention can be changed within a wide range. Let-7a at the same concentration was detected using forward and reverse primers at concentrations ranging from 400 to 600 nM. Prepare a let-7a solution with a concentration of 100pM, and a blank sample (without 1et-7a, that is, the let-7a concentration is 0), and the forward and reverse primer concentrations of the reaction solution are 400nM and 600nM, respectively. Other conditions were operated according to the above steps, and the amplification curves of each reaction solution were measured. As shown in Figure 6, the concentration of forward and reverse primers was in the range of 400-600nM, and the content of 100pM let-7a could be detected. It can be concluded from Figure 6 that the concentration combinations of forward primer and reverse primer are 400nM/400nM, 400nM/600nM, 600nM/400nM, 600nM/600nM, and the ΔCt values corresponding to the detection of 100pM let-7a are 6.73, 6.69, respectively. 5.82, 7.24.

Example 2

The analysis method of miR-141 and miR-141 (5′-UAACACUGUCUGGUAAAGAUGG-3′) was detected by one-step real-time quantitative PCR based on the principle of base stacking hybridization. The detection principle is shown in Figure 7. TaqDNA polymerase and double-stranded DNA binding dye SYBR Green I were used. The functional sequence on the DNA amplification template is the same sequence as the reverse primer (5′-GGCTAAGACAGATGCTC-3′), the target miR-141 binding sequence (5′-CCATCTTTACCAGACAGTGTTA-3′) and the forward primer binding sequence (5′ -TCGCACT-3′).

The following specific implementation of miR-141 detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions or the conditions suggested by the manufacturer. The specific operation steps are as follows: Add 2 μL of reaction buffer solution (50 mM Tris-HCl, 20 mM potassium chloride, 10 mM ammonium sulfate, 2 mM magnesium sulfate, pH 9.0) and 2 μL of DNA amplification template to the eight-tube or 96-well PCR plate well solution (50nM), 2μL forward primer solution (500nM), 2μL reverse primer solution (500nM), 1.6μL dNTPs solution (80μM), 0.2μL Taq DNA polymerase (0.05U/μL), 0.4μL RNase inhibitor ( 0.1U/μL), 0.8 μL SYBR Green I solution (0.4×), 2 μL target miR-141 solution and 7 μL DEPC water. The above reaction solution was placed in a fluorescent quantitative PCR instrument, pre-denatured at 94°C for 30s; denatured at 94°C for 5s, annealed and extended at 60°C for 30s, and a total of 25 cycles were performed. The instrument measured the fluorescence signal of the solution in real time and gave the fluorescence threshold ( C _t ).

Preparation of the working curve: Prepare miR-141 standard solution and blank sample (without miR-141, that is, the miR-141 concentration is 0) of known concentration respectively, follow the above steps to measure the amplification curve of each reaction solution, The fluorescence threshold (C _t ) was derived. Then a standard working curve was made according to the negative logarithmic value of the miR-141 standard solution concentration and its fluorescence threshold (C _t ).

Example 3

An analysis method for detecting miR-21 (5′-UAGCUUAUCAGACUGAUGUUGA-3′) by one-step real-time quantitative PCR based on the principle of base stacking hybridization, the detection principle is shown in Figure 8. TaqDNA polymerase and double-stranded DNA binding dye SYBR Green I were used. The functional sequence on the DNA amplification template is the same sequence as the reverse primer (5′-GGCTAAGACAGATGCTC-3′), the target miR-21 binding sequence (5′-TCAACATCAGTCTGATAAGCTA-3′) and the forward primer binding sequence (5′ -TCGCACT-3′).

The following specific implementation of miR-21 detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions or the conditions suggested by the manufacturer. The specific operation steps are as follows: Add 2 μL of reaction buffer solution (50 mM Tris-HCl, 20 mM potassium chloride, 10 mM ammonium sulfate, 2 mM magnesium sulfate, pH 9.0) and 2 μL of DNA amplification template to the eight-tube or 96-well PCR plate well solution (25nM), 2μL forward primer solution (400nM), 2μL reverse primer solution (600nM), 1.6μL dNTPs solution (500μM), 0.2μL Taq DNA polymerase (0.05U/μL), 0.4μL RNase inhibitor ( 1.0U/μL), 0.8 μL SYBR Green I solution (0.4×), 2 μL target miR-21 solution and 7 μL DEPC water. The above reaction solution was placed in a fluorescent quantitative PCR instrument, pre-denatured at 94°C for 30s; denatured at 94°C for 5s, annealed and extended at 60°C for 30s, and a total of 40 cycles were performed. The instrument measured the fluorescence signal of the solution in real time and gave the fluorescence threshold ( C _t ).

Preparation of the working curve: Prepare miR-21 standard solution and blank sample (without miR-21, that is, the concentration of miR-21 is 0) of known concentration respectively, and follow the above steps to measure the amplification curve of each reaction solution. The fluorescence threshold (C _t ) was derived. Then a standard working curve was made according to the negative logarithmic value of the miR-21 standard solution concentration and its fluorescence threshold (C _t ).

Example 4

An analysis method for detecting miR-200b (5′-UAAUACUGCCUGGUAAUGAUGA-3′) by one-step real-time quantitative PCR based on the principle of base stacking hybridization, the detection principle is shown in Figure 9. TaqDNA polymerase and double-stranded DNA binding dye SYBR Green I were used. The functional sequence on the DNA amplification template is the same sequence as the reverse primer (5′-GGCTAAGACAGATGCTC-3′), the target miR-200b binding sequence (5′-TCATCATTACCAGGCAGTATTA-3′) and the forward primer binding sequence (5′ -TCGCACT-3′).

The following specific implementation of miR-200b detection will further illustrate the present invention. For the experimental methods that do not indicate the specific conditions, usually follow the conventional conditions or the conditions suggested by the manufacturer. The specific operation steps are as follows: Add 2 μL of reaction buffer solution (50 mM Tris-HCl, 20 mM potassium chloride, 10 mM ammonium sulfate, 2 mM magnesium sulfate, pH 9.0) and 2 μL of DNA amplification template to the eight-tube or 96-well PCR plate well solution (200nM), 2μL forward primer solution (800nM), 2μL reverse primer solution (600nM), 1.6μL dNTPs solution (500μM), 0.2μL Taq DNA polymerase (0.08U/μL), 0.4μL RNase inhibitor ( 1.0U/μL), 0.8 μL SYBR Green I solution (0.4×), 2 μL target miR-200b solution and 7 μL DEPC water. The above reaction solution was placed in a fluorescent quantitative PCR instrument, pre-denatured at 94°C for 30s; denatured at 94°C for 5s, annealed and extended at 60°C for 30s, and a total of 30 cycles were performed. The instrument measured the fluorescence signal of the solution in real time and gave the fluorescence threshold ( C _t ).

Preparation of the working curve: Prepare miR-200b standard solution and blank sample (without miR-200b, that is, the concentration of miR-200b is 0) of known concentration respectively, and follow the above steps to measure the amplification curve of each reaction solution. The fluorescence threshold (C _t ) was derived. Then a standard working curve was made according to the negative logarithmic value of the concentration of miR-200b standard solution and its fluorescence threshold (C _t ).

Claims (11)

1. the pcr analysis method of a detection by quantitative microRNA is characterized in that, may further comprise the steps:

Utilize target microRNA after the DNA cloning template is combined, stablized the combination of forward primer and DNA cloning template by the base stacking hybridization, at archaeal dna polymerase effect downward-extension forward primer, with the reverse primer acting in conjunction under, cause the PCR reaction, obtain double-stranded DNA, dyestuff SYBR Green I is combined with double-stranded DNA and is produced fluorescent signal, fluorescence signal intensity in the real time measure reaction system with the standard working curve contrast, calculates the concentration of target microRNA.

2. the pcr analysis method of detection by quantitative microRNA according to claim 1 is characterized in that, described target microRNA is let-7a, miR-141, miR-21, miR-200b.

3. the pcr analysis method of detection by quantitative microRNA according to claim 2 is characterized in that, described let-7a is 5 '-UGAGGUAGUAGGUUGUAUAGUU-3 '; Described DNA cloning template sequence is

5′-GGCTAAGACAGATGCTCTTTGCCAACAGGCCACAGAATTCCTACA

CTCAAAGTCGTACTGAACTATACAACCTACTACCTCATCGCACT-3′。

4. the pcr analysis method of detection by quantitative microRNA according to claim 2 is characterized in that, described miR-141 is 5 '-UAACACUGUCUGGUAAAGAUGG-3 '; Described DNA cloning template sequence is

5′-GGCTAAGACAGATGCTCTTTGCCAACAGGCCACAGAATTCCTACA

CTCAAAGTCGTACTGCCATCTTTACCAGACAGTGTTATCGCACT-3′。

5. the pcr analysis method of detection by quantitative microRNA according to claim 2 is characterized in that, described miR-21 is 5 '-UAGCUUAUCAGACUGAUGUUGA-3 '; Described DNA cloning template sequence is

5′-GGCTAAGACAGATGCTCTTTGCCAACAGGCCACAGAATTCCTACA

CTCAAAGTCGTACTGTCAACATCAGTCTGATAAGCTATCGCACT-3′。

6. the pcr analysis method of detection by quantitative microRNA according to claim 2 is characterized in that, described miR-200b is 5 '-UAAUACUGCCUGGUAAUGAUGA-3 '; Described DNA cloning template sequence is

5′-GGCTAAGACAGATGCTCTTTGCCAACAGGCCACAGAATTCCTACA

CTCAAAGTCGTACTGTCATCATTACCAGGCAGTATTATCGCACT-3′。

7. the pcr analysis method of detection by quantitative microRNA according to claim 1 is characterized in that, described forward primer sequence is 5 '-ATGCGACGATGCGA-3 '.

8. the pcr analysis method of detection by quantitative microRNA according to claim 1 is characterized in that, described archaeal dna polymerase is the Taq archaeal dna polymerase.

9. the pcr analysis method of detection by quantitative microRNA according to claim 1 is characterized in that, described reverse primer sequence is 5 '-GGCTAAGACAGATGCTC-3 '.

10. the pcr analysis method of detection by quantitative microRNA according to claim 1, it is characterized in that, the system of described PCR reaction comprises 50～500 μ M dNTPs, 200～800nM forward primer, 200～800nM reverse primer, 5～200nMDNA amplification template, 0.01～0.08U/ μ L archaeal dna polymerase, dyestuff SYBR Green I, 0.1～1.0U/ μ L RNase inhibitor, target microRNA and DEPC water.

11. the pcr analysis method of detection by quantitative microRNA according to claim 1 is characterized in that, the program of described PCR reaction is: 94 ℃ of denaturation 30s; 94 ℃ of sex change 5s anneal and extension 30s, carry out 20～40 circulations for 55～64 ℃.

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