CN112680509A - Coronary heart disease prognosis evaluation molecular marker miR-302e, reverse transcription primer and amplification primer thereof and application of reverse transcription primer and amplification primer - Google Patents
Coronary heart disease prognosis evaluation molecular marker miR-302e, reverse transcription primer and amplification primer thereof and application of reverse transcription primer and amplification primer Download PDFInfo
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Abstract
The invention discloses a coronary heart disease prognosis evaluation molecular marker miR-302e, a reverse transcription primer, an amplification primer and application thereof, wherein the nucleotide sequence of the coronary heart disease prognosis evaluation molecular marker miR-302e is shown as SEQ ID No.1, the nucleotide sequence of the reverse transcription primer is shown as SEQ ID No.2 in a sequence table, the amplification primer comprises an upstream primer and a downstream primer, the upstream primer and the downstream primer are shown as SEQ ID No.3 and SEQ ID No.4 in the sequence table, and the application of the coronary heart disease prognosis evaluation molecular marker miR-302e in preparing a real-time fluorescent quantitative PCR detection kit for evaluating the coronary heart disease prognosis is disclosed. The miR-302e is used as a new molecular marker for evaluating coronary heart disease prognosis, can evaluate coronary heart disease prognosis, treats patients with coronary heart disease, prolongs the survival time of the patients, improves the survival rate, is a great innovation in coronary heart disease, and has great economic and social benefits.
Description
Technical Field
The invention relates to the technical field of cardiovascular disease molecular biology. In particular to a molecular marker miR-302e for assessing coronary heart disease prognosis, a reverse transcription primer, an amplification primer and application thereof.
Background
At present, cardiovascular diseases (CVD) become the first leading cause of death of urban and rural residents in China, 2016-year statistics shows that the CVD death rate is still the first cause of death of the residents in all the countries, which is higher than that of tumors and other diseases, 45.01% in rural areas and 42.61% in cities, and the CVD morbidity and mortality in China are still in an ascending stage.
Micro RNA (microRNA or miRNA) is a small molecular non-coding RNA consisting of about 22 nucleotides, and after being combined with a 3 'untranslated region (3' UTR) of mRNA generated by transcription of a target gene, the micro RNA can regulate the expression and the function of the gene at the post-transcriptional level, and can be widely involved in various physiological and pathological processes. The research on the expression, function and action mechanism of miRNAs in cardiovascular diseases is a hot point of attention of domestic and foreign scholars in recent years.
Researches show that the micro RNA-302e (miR-302e) is highly expressed in platelets and plasma, mainly participates in the regulation of platelet and endothelial cell functions, plays a very important role in the pathophysiological processes of the occurrence and development of thrombotic CVD, and can be used as a novel biological marker for evaluating platelet activation, antiplatelet drug efficacy and CHD (coronary heart disease) prognosis.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a molecular marker miR-302e for evaluating coronary heart disease prognosis, a reverse transcription primer, an amplification primer and an application thereof, and the problem of preparing a kit for evaluating coronary heart disease prognosis can be effectively solved.
In order to solve the technical problems, the invention provides the following technical scheme:
a molecular marker miR-302e for assessing coronary heart disease prognosis is non-coding RNA, the length of a transcript is 17 nucleotides, and the nucleotide sequence is shown as SEQ ID No.1 in a sequence table.
A reverse transcription primer of a molecular marker miR-302e for evaluating coronary heart disease prognosis is shown as SEQ ID No.2 in a sequence table.
An amplification primer for evaluating a coronary disease prognosis molecular marker miR-302e comprises an upstream primer and a downstream primer, wherein the upstream primer and the downstream primer are shown as SEQ ID No.3 and SEQ ID No.4 in a sequence table.
The application of the molecular marker miR-302e for assessing coronary heart disease prognosis in the preparation of a real-time fluorescent quantitative PCR detection kit for assessing coronary heart disease prognosis.
The application of the molecular marker miR-302e for assessing coronary heart disease prognosis in the preparation of the real-time fluorescent quantitative PCR detection kit for assessing coronary heart disease prognosis comprises the following steps: the reverse transcription primer of the coronary heart disease prognosis molecular marker miR-302e, the specific amplification primer and the specific amplification primer sequence expressed by the internal reference gene U6 are shown in the sequence table SEQ ID No.5 and SEQ ID No.6 as the upstream primer and the downstream primer sequences, and the reagent for extracting miRNA from blood plasma and carrying out reverse transcription and fluorescence quantitative PCR.
The application of the molecular marker miR-302e for assessing coronary heart disease prognosis in the preparation of the real-time fluorescent quantitative PCR detection kit for assessing coronary heart disease prognosis comprises the following steps:
(1) reagent for extracting total RNA from coronary heart disease or healthy people: comprises 20mL of Trizol liquid, 80mL of chloroform, 80mL of isopropanol, 40mL of RNA degradation inhibiting solvent, 30mL of ethanol with volume fraction of 75 percent and 10mL of DEPC water;
(2) reagents for reverse transcription using total RNA as template: the method comprises the following steps of evaluating a reverse transcription primer of a coronary heart disease prognostic molecular marker miR-302 e: the concentration is 10 muM, 30 muL; downstream primer of reference gene U6: the concentration is 10 muM, 30 muL; reverse transcription Buffer 5 × RT Buffer 150 μ L; the concentration of the reverse transcriptase M-MLV is 200U/. mu.L, and 50. mu.L;
(3) real-time quantitative PCR of cDNA: comprises miRNA-302e real-time fluorescent quantitative PCR specific primers: the concentration is 3 muM, and each 25 muL; specific amplification primers for expression of reference gene U6: the concentration is 3 muM, and each 25 muL; real-time fluorescent quantitative SYBR dye SYBR Green qPCR Mix500 μ L; RNase free water 25. mu.L.
The application of the molecular marker miR-302e for assessing coronary heart disease prognosis in the preparation of the real-time fluorescent quantitative PCR detection kit for assessing coronary heart disease prognosis comprises the following steps:
(1) extracting plasma RNA;
(2) reverse transcription of miRNA-302e RNA;
(3) carrying out real-time quantitative PCR by using a specific primer of miRNA-302 e;
(4) and (3) analyzing data of miRNA-302e expression quantity: a relative quantification method 2^ -delta-Delta Ct method is adopted.
The application of the molecular marker miR-302e for assessing coronary heart disease prognosis in the preparation of the real-time fluorescence quantitative PCR detection kit for assessing coronary heart disease prognosis is as follows in step (1):
(1-1) adding 100 mu L of blood plasma into an EP tube already containing 1ml of Trizol liquid, and fully and uniformly mixing;
(1-2) standing at room temperature for 5 minutes, then adding 200. mu.L of chloroform, covering the EP tube and shaking vigorously for 30 seconds, and then centrifuging at 12000 rpm for 10 minutes;
(1-3) taking the upper aqueous phase in a new EP tube, adding 500 mu L of isopropanol, and mixing by mild inversion; standing at room temperature for 10 minutes, and centrifuging at 12000 rpm for 10 minutes;
(1-4) carefully discarding the supernatant, adding 1mL of 75% ethanol by volume, mixing by vortexing, and centrifuging at 12000 rpm for 5 minutes at 4 ℃; repeating the operation once;
(1-5) discarding the supernatant, and drying at room temperature or in vacuum for 5-10 minutes; dissolve the RNA with 50. mu.L DEPC water;
(1-6) determination of RNA concentration: the assay was performed using a nucleic acid concentration meter, and 1. mu.L of the RNA sample was pipetted into the sample wells and the RNA concentration was directly determined from the readings. The ratio of OD260/OD280 measured by a nucleic acid concentration measuring instrument is 1.8-2.0, and the RNA purity is considered to be good; finally, the RNA was stored in a freezer at-80 ℃ until use.
In the step (2), the reverse transcription of miRNA-302e RNA is carried out by using a reverse transcription kit of Shanghai Biyun biotechnology, Inc.:
(2-1) removal of genomic DNA: unfreezing a template Total RNA and a 5 XgDNA Eraser Buffer on ice, unfreezing the 5 XRT Buffer and DEPC water at the temperature of 15-25 ℃, and quickly placing on ice after unfreezing; each solution was mixed well and centrifuged briefly before use to allow all liquid to settle to the bottom of the tube; denaturation of RNA, namely, after the RNA sample is thermally denatured at 65 ℃ for 5 minutes, immediately placing the RNA sample in ice water for cooling; adding 0.5 mu g of Total RNA, 2 mu L of 5 XgDNA Eraser Buffer and DEPC-treated Water to the Total volume of 10 mu to prepare a reaction mixed solution on ice, and finally adding an RNA sample;
(2-2) incubating at 37 ℃ for 2 minutes on a PCR instrument or in a water bath; quickly placing on ice for later use;
(2-3) reverse transcription system preparation: preparing a reaction solution on ice, and performing reverse transcription immediately after the reaction solution is mixed gently and uniformly; the reverse transcription system comprises: 4 μ L of 5 × RT Buffer; a target gene miRNA-302e reverse transcription primer or a reference gene U6 downstream primer with the concentration of 10 mu M and the volume of 1 mu L; 2 μ L of BeyoRTTMII M-MLV reverse transcriptase; 3 μ L of DEPC-treated water; 10 μ L of degenomic RNA;
(2-4) incubation at 42 ℃ for 60 minutes to perform reverse transcription reaction, followed by incubation at 80 ℃ for 10 minutes to inactivate reverse transcriptase and then placing on ice;
and (2-5) the obtained cDNA can be used for subsequent real-time fluorescence quantitative PCR immediately or after being frozen at-80 ℃, and the cDNA is preferably prevented from excessive repeated freezing and thawing.
The application of the molecular marker miR-302e for assessing coronary heart disease prognosis in the preparation of the real-time fluorescence quantitative PCR detection kit for assessing coronary heart disease prognosis is as follows in step (3):
(3-1) melting and mixing various solutions required for PCR reaction, BeyofastTMSYBR Green qPCR Mix is completely melted and evenly mixed and then placed in an ice box;
(3-2) setting a PCR reaction system on an ice bath, wherein the PCR reaction system comprises: beyofastTMThe SYBR Green qPCR Mix is 10. mu.L, and the concentration of the upstream primer and the downstream primer is 1. mu.L respectively at 3. mu.M; the cDNA is 2 mu L, and the content is 1-10 ng; RNase free water was 6. mu.L;
(3-3) lightly beating and uniformly mixing the mixture by using a pipettor or slightly mixing the mixture by Vortex, and centrifuging the mixture for a plurality of seconds at room temperature to ensure that the liquid is accumulated at the bottom of the tube;
(3-4) placing the set PCR reaction tube or PCR reaction plate on a fluorescent quantitative PCR instrument to start PCR reaction; the procedure for the PCR reaction was:
a. pre-denaturation: 2 minutes at 95 ℃; b. denaturation: 15 seconds at 95 ℃; c. annealing/extending: 30 seconds at 60 ℃; d. repeating steps b and c for a total of 40 cycles; e. and (3) melting curve analysis: 15 seconds at 95 ℃, 15 seconds at 60 ℃ and 15 seconds at 95 ℃; f. results were analyzed using software provided with a fluorescent quantitative PCR instrument.
The technical scheme of the invention achieves the following beneficial technical effects:
the miR-302e is a non-coding RNA and can be effectively used for preparing a kit for evaluating the prognosis of coronary heart disease, and the miR-302e is used as a new molecular marker for evaluating the prognosis of coronary heart disease, can evaluate the prognosis of coronary heart disease in time, treats patients with coronary heart disease in time, prolongs the survival time of the patients and improves the survival rate, so that the miR-302e is a great innovation on the coronary heart disease and has great economic and social benefits.
Research proves that the miRNA-302e is expressed and reduced in coronary heart disease, and the recurrence rate of patients with coronary heart disease with low miRNA-302e expression is higher. Therefore, by detecting the expression level of miRNA-302e in the plasma of a coronary heart disease patient, the method can be used for carrying out prognosis examination and diagnosis on the coronary heart disease patient, the accuracy is up to more than 93%, timely treatment is carried out, the recurrence rate and the survival quality of the patient are reduced, and the method has profound clinical application significance, popularization and application values and great economic and social benefits.
Drawings
FIG. 1 is a graph of the difference of miR-302e expression in blood plasma of healthy patients and patients with coronary heart disease by real-time fluorescence quantitative PCR analysis according to the invention;
FIG. 2 is a graph showing the difference between the low-expression miR-302e group and the high-expression miR-302e group in the plasma of a coronary heart disease patient in the recurrence rate analysis by real-time fluorescent quantitative PCR analysis of the invention.
Detailed Description
Example 1 evaluation of coronary artery disease prognostic molecular marker miR-302e
The coronary heart disease prognosis evaluation molecular marker miR-302e is non-coding RNA, the length of a transcript is 17 nucleotides, and the nucleotide sequence is shown as SEQ ID No.1 in a sequence table.
SEQ ID No.1：UAAGUGCUUCCAUGCUU。
Embodiment 2, application of the molecular marker miR-302e for assessing coronary heart disease prognosis in preparation of a real-time fluorescence quantitative PCR detection kit for assessing coronary heart disease prognosis.
The real-time fluorescence quantitative PCR detection kit for assessing coronary heart disease prognosis comprises: the reverse transcription primer of the molecular marker miR-302e for assessing coronary heart disease prognosis has a sequence of SEQ ID No.2 and a specific amplification primer, the primer sequences of the specific amplification primer are SEQ ID No.3 and SEQ ID No.4, the specific amplification primer sequence expressed by the internal reference gene U6 is shown, the upstream primer and the downstream primer sequences of the specific amplification primer are SEQ ID No.5 and SEQ ID No.6 in a sequence table, and the reagent for extracting miRNA from blood plasma and performing reverse transcription and fluorescence quantitative PCR.
Reverse transcription primer, SEQ ID No. 2:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAGCATGGAAGCACTTA
specific amplification primers, an upstream primer SEQ ID No. 3:
TAAGTGCTTCCATGCTTGTCGTA。
specific amplification primers, downstream primers SEQ ID No. 4:
AGTGCAGGGTCCGAGGTATT。
reference gene U6, upstream primer SEQ ID No. 5: CTCGCTTCGGCAGCACA are provided.
Reference gene U6, downstream primer SEQ ID No. 6: AACGCTTCACGAATTTGCGT are provided.
The real-time fluorescence quantitative PCR detection kit for assessing coronary heart disease prognosis comprises the following components (used for 30 reactions):
(1) reagent for extracting total RNA from coronary heart disease or healthy people: comprises 20mL of Trizol liquid, 80mL of chloroform, 80mL of isopropanol, 40mL of RNA degradation inhibiting solvent, 30mL of ethanol with volume fraction of 75 percent and 10mL of DEPC water;
(2) reagents for reverse transcription using total RNA as template: the method comprises the following steps of evaluating a reverse transcription primer of a coronary heart disease prognostic molecular marker miR-302 e: the concentration is 10 muM, 30 muL; downstream primer of reference gene U6: the concentration is 10 muM, 30 muL; reverse transcription Buffer 5 × RT Buffer 150 μ L; reverse transcriptase M-MLV 50. mu.L;
(3) real-time quantitative PCR of cDNA: comprises miRNA-302e real-time fluorescent quantitative PCR specific primers: the concentration is 3 muM, and each 25 muL; specific amplification primers for expression of reference gene U6: the concentration is 3 muM, and each 25 muL; real-time fluorescent quantitative SYBR dye SYBR Green qPCR Mix500 μ L; RNase free water 25. mu.L.
The detection method of the plasma sample miRNA-302e comprises the following steps:
1. collecting blood plasma of patients with coronary heart disease or healthy patients to be detected, putting the blood plasma into a freezing storage tube filled with the RNA degradation inhibiting solvent, and putting the freezing storage tube into a refrigerator at the temperature of minus 80 ℃ for later use.
2. Extraction of plasma RNA:
(1) 100 μ L of plasma was added to an EP tube already containing 1ml of Trizol solution and mixed well.
(2) After 5 minutes at room temperature, 200. mu.L of chloroform was added, the EP tube was closed and shaken vigorously for 30 seconds. Centrifuge at 12000 rpm for 10 minutes.
(3) The upper aqueous phase was taken in a new EP tube (without intermediate precipitate and underflow mixing in), 500. mu.L of isopropanol was added and mixed by gentle inversion. The mixture was left at room temperature for 10 minutes and centrifuged at 12000 rpm for 10 minutes.
(4) The supernatant was carefully discarded, 1mL of 75% ethanol was added, vortexed, and centrifuged at 12000 rpm for 5 minutes at 4 ℃. The operation was repeated once.
(5) The supernatant is discarded (the residual liquid is removed as much as possible), and dried at room temperature or under vacuum for 5-10 minutes (taking care not to dry too much, otherwise the solubility of RNA is reduced). The RNA was dissolved with 50. mu.L of DEPC water.
(6) Determination of RNA concentration: the assay was performed using a nucleic acid concentration meter, and 1. mu.L of the RNA sample was pipetted into the sample wells and the RNA concentration was directly determined from the readings. The ratio of OD260/OD280 measured by the nucleic acid concentration measuring instrument is 1.8-2.0, and the RNA purity is considered to be good. Finally, the RNA was stored in a freezer at-80 ℃ until use.
3. Reverse transcription of miRNA-302e RNA: a reverse transcription kit (D7170S) from Shanghai Biyuntian Biotechnology Ltd was used. The method comprises the following specific steps.
(1) Removal of genomic DNA: unfreezing a template Total RNA and a template 5 XgDNA Eraser Buffer on ice, unfreezing the template 5 XRT Buffer and the template DEPC-treated Water at room temperature (15-25 ℃), and quickly placing the unfrozen template on ice. Each solution was mixed well and centrifuged briefly before use to allow all liquid to settle to the bottom of the tube. Denaturation of RNA, the RNA sample was heat-denatured at 65 ℃ for 5 minutes, and then immediately cooled in ice water. The following ingredients were mixed on ice to prepare a reaction mixture, which was then dispensed into each reaction tube, and finally the RNA sample was added.
TABLE 1
| Reagent | Amount of the composition used |
| 5×gDNA Eraser Buffer | 2μL |
| Total RNA | up to 0.5μg |
| DEPC-treated Water | To 10 μ L |
| Total volume | 10μL |
(2) Incubate for 2 minutes at 37 ℃ on a PCR instrument or in a water bath. Quickly placing on ice for standby.
(3) Preparing a reverse transcription system: the reaction solution was prepared on ice, and after gentle mixing, reverse transcription was immediately performed. The mixture was prepared according to the reverse transcription reaction system shown in the following table.
TABLE 2
(4) The reverse transcription reaction was performed by incubating at 42 ℃ for 60 minutes, followed by incubation at 80 ℃ for 10 minutes to inactivate the reverse transcriptase and then placing on ice. For templates with complex secondary structures or high GC content, the reverse transcription temperature can be increased to 50 ℃ to enhance the reverse transcription efficiency.
(5) The obtained cDNA can be used for subsequent real-time fluorescent quantitative PCR immediately or after being frozen at-80 ℃, and the cDNA is suitable for avoiding excessive repeated freezing and thawing.
4. Carrying out real-time quantitative PCR by using a specific primer of miRNA-302 e: the specific primers are synthesized by the biological engineering (Shanghai) corporation, and comprise specific primers for detecting miRNA-302e expression, wherein the primer sequences are SEQ ID NO.3 and SEQ ID NO.4, and the primer sequences expressed by an internal reference gene U6 are SEQ ID NO.5 and SEQ ID NO. 6. Real-time quantitative PCR other reagents Beyofast of Shanghai Biyuntian biotechnology limitedTMSYBR Green qPCR Mix (2X), the procedure is as follows.
(1) Various solutions required for the PCR reaction, Beyofast, were thawed and mixed wellTMSYBR Green qPCR Mix was completely thawed and placed in an ice box after mixing.
(2) The PCR reaction system (in 96-well plates for example) was set up in an ice bath as shown in Table 3; and is shown in Table 4 with reference to the reference gene U6.
TABLE 3
TABLE 4
(3) In general, the amount of the DNA template is 1 to 10ng of cDNA as a reference amount, and a good detection effect can be obtained when the final concentration of the primer is 0.2 to 0.5. mu.M, or the final concentration of the primer can be adjusted according to circumstances. If necessary, the template may be subjected to a gradient dilution to determine the optimal template usage. When cDNA obtained by reverse transcription PCR reaction is directly used as a template, the amount of cDNA added is not more than 10% of the total volume of the PCR reaction. The recommended reaction system of the 96-well plate is 20 mu L, and the reaction system can be proportionally expanded or reduced according to the actual experiment requirements.
(4) Gently pipetting the mixture or gently Vortex mixing the mixture, and centrifuging the mixture at room temperature for several seconds to accumulate the liquid at the bottom of the tube.
(5) And placing the set PCR reaction tube or PCR reaction plate on a fluorescent quantitative PCR instrument to start PCR reaction.
(6) PCR reaction procedure: pre-denaturation of the template was performed prior to real-time fluorescent quantitative PCR reaction, typically set at 95 ℃ for 2 minutes. The following PCR program was used, which is exemplified by a BerleCFX 96 fluorescent quantitative PCR instrument:
a. pre-denaturation: 95 ℃ for 2 minutes
b. Denaturation: 95 ℃ for 15 seconds
c. Annealing/extending: 60 ℃ for 30 seconds
d. Repeating steps b and c for a total of 40 cycles
e. Melting curve analysis (optional): 95 ℃ for 15 seconds, 60 ℃ for 15 seconds, 95 ℃ for 15 seconds
f. Analysis of results Using software supplied by fluorescent quantitative PCR Instrument
The three-step process requires only one additional step of 72 c 30 seconds after annealing/extension, followed by 40 cycles of repeating steps b and c and the additional step.
5. And (3) analyzing data of miRNA-302e expression quantity:
the experimental data were included in the plasma of 60 patients with coronary heart disease and 60 healthy patients. The result analysis of real-time quantitative PCR adopts a relative quantitative method, namely a 2^ -delta Delta Ct method. The method comprises the following specific steps:
firstly, the Ct values of all genes in one experiment are well organized, and then the Ct value of the target gene miRNA-302e in each group of plasma samples is subtracted by the Ct value of the self-reference gene U6 to obtain a number which is delta Ct; the conversion formula is: Δ Ct ═ Ct (target gene miRNA-302e) -Ct (reference gene U6);
then, the delta Ct of each target gene miRNA-302e of each group of plasma samples is calculated. And (3) subtracting the delta Ct of the plasma sample of the healthy group from the delta Ct of the plasma sample of the coronary heart disease patient in the experiment, and simultaneously taking the opposite numbers of all the results, wherein the result obtained by the operation of the step is delta Ct.
Finally, the power of 2 is performed on- Δ Ct, i.e., 2^ Δ Ct gives the fold change of expression. Three replicates were performed and statistical analysis was performed using a non-parametric t-test. The expression level of miRNA-302e in the plasma of patients with coronary heart disease is lower than that of healthy group (see figure 1), and the difference is statistically significant (p < 0.05).
6. The follow-up statistics of 60 coronary heart disease patients included in the above experiments include the time of first onset, treatment condition, recurrence status, etc. of the patients, and the follow-up time is at least 12 months. Selecting the expression value of the fluorescent real-time quantitative PCR analysis from the selected coronary heart disease patients as a reference standard, and comparing the obtained result with the healthy patients. The miRNA-302e expression in the plasma of the coronary heart disease patient is lower than that in the plasma of a healthy person and is defined as a miRNA-302e low expression group, and the miRNA-302e expression in the plasma of the coronary heart disease patient is higher than that in the healthy person and is defined as a high expression group. Through analysis, the recurrence number of miRNA-302e low-expression patients is obviously higher than that of miRNA-302e high-expression patients
(see FIG. 2), the differences were statistically significant (p < 0.05). Therefore, the miRNA-302e can be used as a specific molecular marker for the prognosis of patients with coronary heart disease.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications are possible which remain within the scope of the appended claims.
Sequence listing
<110> Henan province Hospital of traditional Chinese medicine (second subsidiary Hospital of Henan university of traditional Chinese medicine)
<120> molecular marker miR-302e for assessing coronary heart disease prognosis, reverse transcription primer, amplification primer and application thereof
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Claims (10)
1. A molecular marker miR-302e for evaluating coronary heart disease prognosis is characterized in that the molecular marker is non-coding RNA, the length of a transcript is 17 nucleotides, and the nucleotide sequence is shown as SEQ ID No.1 in a sequence table.
2. A reverse transcription primer for evaluating a coronary heart disease prognostic molecular marker miR-302e is characterized in that the nucleotide sequence of the reverse transcription primer is shown as SEQ ID No.2 in a sequence table.
3. An amplification primer for evaluating a coronary disease prognostic molecular marker miR-302e is characterized by comprising an upstream primer and a downstream primer, wherein the upstream primer and the downstream primer are shown as SEQ ID No.3 and SEQ ID No.4 in a sequence table.
4. The application of the molecular marker miR-302e for assessing coronary heart disease prognosis in the preparation of a real-time fluorescence quantitative PCR detection kit for assessing coronary heart disease prognosis according to claim 1.
5. The application of the molecular marker miR-302e for assessing coronary heart disease prognosis in the preparation of the real-time fluorescence quantitative PCR detection kit for assessing coronary heart disease prognosis according to claim 4, wherein the real-time fluorescence quantitative PCR detection kit for assessing coronary heart disease prognosis comprises: the reverse transcription primer of the molecular marker miR-302e for evaluating coronary heart disease prognosis in claim 2, the specific amplification primer in claim 3 and the specific amplification primer sequence expressed by the internal reference gene U6, wherein the upstream primer and the downstream primer sequences are SEQ ID No.5 and SEQ ID No.6 in the sequence table, and the reagent for extracting miRNA from blood plasma and carrying out reverse transcription and fluorescence quantitative PCR.
6. The application of the molecular marker miR-302e for assessing coronary heart disease prognosis in the preparation of the real-time fluorescence quantitative PCR detection kit for assessing coronary heart disease prognosis according to claim 5, wherein the real-time fluorescence quantitative PCR detection kit for assessing coronary heart disease prognosis comprises:
(1) reagent for extracting total RNA from coronary heart disease or healthy people: comprises 20mL of Trizol liquid, 80mL of chloroform, 80mL of isopropanol, 40mL of RNA degradation inhibiting solvent, 30mL of ethanol with volume fraction of 75 percent and 10mL of DEPC water;
(2) reagents for reverse transcription using total RNA as template: the method comprises the following steps of evaluating a reverse transcription primer of a coronary heart disease prognostic molecular marker miR-302 e: the concentration is 10 muM, 30 muL; downstream primer of reference gene U6: the concentration is 10 muM, 30 muL; reverse transcription Buffer 5 × RT Buffer 150 μ L; the concentration of the reverse transcriptase M-MLV is 200U/. mu.L, and 50. mu.L;
(3) real-time quantitative PCR of cDNA: comprises miRNA-302e real-time fluorescent quantitative PCR specific primers: the concentration is 3 muM, and each 25 muL; specific amplification primers for expression of reference gene U6: the concentration is 3 muM, and each 25 muL; real-time fluorescent quantitative SYBR dye SYBR Green qPCR Mix500 μ L; RNase free water 25. mu.L.
7. The application of the coronary heart disease prognosis evaluation molecular marker miR-302e in the preparation of the coronary heart disease prognosis evaluation real-time fluorescence quantitative PCR detection kit according to claim 5 is characterized in that the detection method of the coronary heart disease prognosis evaluation real-time fluorescence quantitative PCR detection kit comprises the following steps:
(1) extracting plasma RNA;
(2) reverse transcription of miRNA-302e RNA;
(3) carrying out real-time quantitative PCR by using a specific primer of miRNA-302 e;
(4) and (3) analyzing data of miRNA-302e expression quantity: a relative quantification method 2^ -delta-Delta Ct method is adopted.
8. The application of the molecular marker miR-302e for assessing coronary heart disease prognosis in the preparation of the real-time fluorescence quantitative PCR detection kit for assessing coronary heart disease prognosis according to claim 7 is characterized in that in the step (1):
(1-1) adding 100 mu L of blood plasma into an EP tube already containing 1ml of Trizol liquid, and fully and uniformly mixing;
(1-2) standing at room temperature for 5 minutes, then adding 200. mu.L of chloroform, covering the EP tube and shaking vigorously for 30 seconds, and then centrifuging at 12000 rpm for 10 minutes;
(1-3) taking the upper aqueous phase in a new EP tube, adding 500 mu L of isopropanol, and mixing by mild inversion; standing at room temperature for 10 minutes, and centrifuging at 12000 rpm for 10 minutes;
(1-4) carefully discarding the supernatant, adding 1mL of 75% ethanol by volume, mixing by vortexing, and centrifuging at 12000 rpm for 5 minutes at 4 ℃; repeating the operation once;
(1-5) discarding the supernatant, and drying at room temperature or in vacuum for 5-10 minutes; dissolve the RNA with 50. mu.L DEPC water;
(1-6) determination of RNA concentration: the assay was performed using a nucleic acid concentration meter, and 1. mu.L of the RNA sample was pipetted into the sample wells and the RNA concentration was directly determined from the readings. The ratio of OD260/OD280 measured by a nucleic acid concentration measuring instrument is 1.8-2.0, and the RNA purity is considered to be good; finally, the RNA was stored in a freezer at-80 ℃ until use.
9. The application of the molecular marker miR-302e for coronary heart disease prognosis evaluation in the preparation of the real-time fluorescence quantitative PCR detection kit for coronary heart disease prognosis evaluation according to claim 8, wherein in the step (2), the reverse transcription of miRNA-302e RNA is performed by using a reverse transcription kit of Shanghai Bin Yuntan biotechnology, Inc.:
(2-1) removal of genomic DNA: unfreezing a template Total RNA and a 5 XgDNA Eraser Buffer on ice, unfreezing the 5 XRT Buffer and DEPC water at the temperature of 15-25 ℃, and quickly placing on ice after unfreezing; each solution was mixed well and centrifuged briefly before use to allow all liquid to settle to the bottom of the tube; denaturation of RNA, namely, after the RNA sample is thermally denatured at 65 ℃ for 5 minutes, immediately placing the RNA sample in ice water for cooling; adding 0.5 mu g of Total RNA, 2 mu L of 5 XgDNA Eraser Buffer and DEPC-treated Water to the Total volume of 10 mu to prepare a reaction mixed solution on ice, and finally adding an RNA sample;
(2-2) incubating at 37 ℃ for 2 minutes on a PCR instrument or in a water bath; quickly placing on ice for later use;
(2-3) reverse transcription system preparation: preparing a reaction solution on ice, and performing reverse transcription immediately after the reaction solution is mixed gently and uniformly; the reverse transcription system comprises: 4 μ L of 5 × RT Buffer; a target gene miRNA-302e reverse transcription primer or a reference gene U6 downstream primer with the concentration of 10 mu M and the volume of 1 mu L; 2 μ L of BeyoRTTMII M-MLV reverse transcriptase; 3 μ L of DEPC-treated water; 10 μ L of degenomic RNA;
(2-4) incubation at 42 ℃ for 60 minutes to perform reverse transcription reaction, followed by incubation at 80 ℃ for 10 minutes to inactivate reverse transcriptase and then placing on ice;
and (2-5) the obtained cDNA can be used for subsequent real-time fluorescence quantitative PCR immediately or after being frozen at-80 ℃, and the cDNA is preferably prevented from excessive repeated freezing and thawing.
10. The application of the molecular marker miR-302e for assessing coronary heart disease prognosis in the preparation of the real-time fluorescence quantitative PCR detection kit for assessing coronary heart disease prognosis according to claim 9 is characterized in that in the step (3):
(3-1) melting and mixing various solutions required for PCR reaction, BeyofastTMSYBR Green qPCR Mix is completely melted and evenly mixed and then placed in an ice box;
(3-2) setting a PCR reaction system on an ice bath, wherein the PCR reaction system comprises: beyofastTMThe SYBR Green qPCR Mix is 10. mu.L, and the concentration of the upstream primer and the downstream primer is 1. mu.L respectively at 3. mu.M; the cDNA is 2 mu L, and the content is 1-10 ng; RNase free water was 6. mu.L;
(3-3) lightly beating and uniformly mixing the mixture by using a pipettor or slightly mixing the mixture by Vortex, and centrifuging the mixture for a plurality of seconds at room temperature to ensure that the liquid is accumulated at the bottom of the tube;
(3-4) placing the set PCR reaction tube or PCR reaction plate on a fluorescent quantitative PCR instrument to start PCR reaction; the procedure for the PCR reaction was:
a. pre-denaturation: 2 minutes at 95 ℃; b. denaturation: 15 seconds at 95 ℃; c. annealing/extending: 30 seconds at 60 ℃; d. repeating steps b and c for a total of 40 cycles; e. and (3) melting curve analysis: 15 seconds at 95 ℃, 15 seconds at 60 ℃ and 15 seconds at 95 ℃; f. results were analyzed using software provided with a fluorescent quantitative PCR instrument.
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