CN107858419A - MiRNA application, product and detection method using it - Google Patents
MiRNA application, product and detection method using it Download PDFInfo
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- CN107858419A CN107858419A CN201711105641.3A CN201711105641A CN107858419A CN 107858419 A CN107858419 A CN 107858419A CN 201711105641 A CN201711105641 A CN 201711105641A CN 107858419 A CN107858419 A CN 107858419A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention provides a kind of miRNA application, product and detection method using it, it is related to biological technical field, miR 21 5p provided by the invention, miR 30a 3p or miR 30a 5p are found through experiments that to express in the cardiac muscle cell with heart disease and be remarkably decreased.Therefore miR 21 5p, the miR 30a 3p or miR 30a 5p provided in the present invention can be estimated and detect as the risk of Heart Failure associated diseases.In addition, present invention also offers above-mentioned miR 21 5p, miR 30a 3p or miR 30a 5p detection method, Microrna change is counted by means of the technology of real-time quantitative PCR, has the advantages that analysis sample requirement is small, automaticity is high, avoid pollution.
Description
Technical field
The present invention relates to biological technical field, more particularly, to a kind of miRNA application, using its product and detection side
Method.
Background technology
At present, angiocardiopathy is to cause the first killer of human death, and heart failure is one kind in angiocardiopathy,
Heart failure is not an independent disease, but stage end last of heart disease development, but heart failure colony in recent years
Rejuvenation trend is fairly obvious, but does not cause the enough attention of young people.How efficient diagnosis and prevention heart failure
Medical science and biological study urgent problem to be solved at present.
Microrna (microRNA, miRNA) is the non-protein that one kind is widely present in eucaryote, length is 21-25nt
Matter encodes tiny RNA, and they can be expressed with sequence-specific fashion regulatory gene, in development, apoptosis, metabolism and human diseases side
Face all plays a part of can not be ignored.MiRNA physiological and pathological regulatory mechanism is that one be highly valued in recent years is new
Type subject.
Recent studies indicate that angiocardiopathy can cause the expression quantity of Microrna special in vivo to occur significantly
Change, therefore, the change by detecting the specific Microrna in body fluid can play a part of diagnosis, prevention of cardiovascular disease.
But at present to the research still Shortcomings of the related specific Microrna of heart failure.
In view of this, it is special to propose the present invention.
The content of the invention
First purpose of the present invention be to provide miR-21-5p, miR-30a-3p or miR-30a-5p any one or
Application of many kinds of substance in the product for Diagnosing Cardiac disease is prepared, to alleviate present in prior art to heart disease
The technical problem that related specific tiny RNA research still has some deficits.
Second object of the present invention is to provide a kind of reagent for Diagnosing Cardiac disease.
Third object of the present invention is to provide a kind of kit for Diagnosing Cardiac disease.
Fourth object of the present invention be to provide miR-21-5p, miR-30a-3p or miR-30a-5p any one or
The detection method of many kinds of substance, asked with specific not strong enough the technology of detection alleviated in the prior art to above-mentioned three kinds of tiny RNAs
Topic.
Following any one or more material of (a)-(c) provided by the invention is preparing the product for Diagnosing Cardiac disease
In application：
(a)miR-21-5p；
(b)miR-30a-3p；
(c)miR-30a-5p。
Further, the nucleotide sequence of the miR-21-5p is as shown in SEQ ID No.1, the miR-30a-3p's
Nucleotide sequence is as shown in SEQ ID No.2, and the nucleotide sequence of the miR-30a-5p is as shown in SEQ ID No.3.
Further, the product is reagent or kit.
Further, the heart disease is heart failure.
Present invention also offers a kind of reagent for Diagnosing Cardiac disease, it is any that the reagent includes following (A)-(C)
One or more materials：
(A) it is used for the primer for detecting miR-21-5p；
(B) it is used for the primer for detecting miR-30a-3p；
(C) it is used for the primer for detecting miR-30a-5p.
Present invention also offers a kind of kit for Diagnosing Cardiac disease, the kit includes following (A)-(C)
Any one or more material or above-mentioned reagent：
(A) it is used for the primer for detecting miR-21-5p；
(B) it is used for the primer for detecting miR-30a-3p；
(C) it is used for the primer for detecting miR-30a-5p.
Further, there is the primer for being used to detect miR-21-5p the upstream of sequence as shown in SEQ ID No.4 to draw
Thing and as shown in SEQ ID No.5 sequence anti-sense primer；
The primer for being used to detect miR-30a-3p is with the sense primer of sequence as shown in SEQ ID No.6 and such as
The anti-sense primer of sequence shown in SEQ ID No.7；
The primer for being used to detect miR-30a-5p is with the sense primer of sequence as shown in SEQ ID No.8 and such as
The anti-sense primer of sequence shown in SEQ ID No.9.
Further, the heart disease is heart failure.
In addition, present invention also offers a kind of detection method of any one or more material of following (a)-(c), detected
The method of stating includes：Extract sample rna and carry out fluorescent quantitation qPCR reactions, obtain any one or more material of (a)-(c)
Relative expression quantity；
(a)-(c) be：
(a)miR-21-5p；
(b)miR-30a-3p；
(c)miR-30a-5p。
Further, sample rna is extracted using Trizol methods.
MiR-21-5p, miR-30a-3p or miR-30a-5p provided by the invention are found through experiments that with heart disease
Express and be remarkably decreased in the cardiac muscle cell of disease.Therefore miR-21-5p, miR-30a-3p or the miR-30a-5p provided in the present invention
It can estimate and detect as the risk of Heart Failure associated diseases.In addition, present invention also offers above-mentioned miR-21-5p,
MiR-30a-3p or miR-30a-5p detection method, Microrna change, tool are counted by means of the technology of real-time quantitative PCR
Have the advantages that analysis sample requirement is small, automaticity is high, avoid pollution.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art
The required accompanying drawing used is briefly described in embodiment or description of the prior art, it should be apparent that, in describing below
Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid
Put, other accompanying drawings can also be obtained according to these accompanying drawings.
Figure 1A detects miR-21-5p in heart failure disease to be provided in an embodiment of the present invention using fluorescence quantifying PCR method
People and the testing result figure in check sample；
Figure 1B detects miR-30a-3p in heart failure disease to be provided in an embodiment of the present invention using fluorescence quantifying PCR method
People and the testing result figure in check sample；
Fig. 1 C detect miR-30a-5p in heart failure disease to be provided in an embodiment of the present invention using fluorescence quantifying PCR method
People and the testing result figure in check sample；
Fig. 2A is the inspection provided in an embodiment of the present invention to detection miR-21-5p in heart failure patient and check sample
Survey the ROC analysis result figures of result；
Fig. 2 B are to be provided in an embodiment of the present invention to detecting miR-30a-3p in heart failure patient and check sample
The ROC analysis result figures of testing result；
Fig. 2 C are to be provided in an embodiment of the present invention to detecting miR-30a-5p in heart failure patient and check sample
The ROC analysis result figures of testing result；
Fig. 3 A are provided in an embodiment of the present invention related in sample is detected to miR-30a-3p to detection miR-21-5p
Property analysis result figure；
Fig. 3 B are provided in an embodiment of the present invention related in sample is detected to miR-30a-5p to detection miR-21-5p
Property analysis result figure；
Fig. 3 C are the phase provided in an embodiment of the present invention to detection miR-30a-3p and miR-30a-5p in sample is detected
Closing property analysis result figure；
Fig. 4 A are detecting sample to be provided in an embodiment of the present invention to detection miR-21-5p and clinical examination NT-proBNP
In correlation analysis result figure；
Fig. 4 B are detecting sample to be provided in an embodiment of the present invention to detection miR-30a-3p and clinical examination NT-proBNP
In correlation analysis result figure；
Fig. 4 C are detecting sample to be provided in an embodiment of the present invention to detection miR-30a-5p and clinical examination NT-proBNP
In correlation analysis result figure.
Embodiment
Technical scheme is clearly and completely described below in conjunction with accompanying drawing, it is clear that described implementation
Example is part of the embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, ordinary skill
The every other embodiment that personnel are obtained under the premise of creative work is not made, belongs to the scope of protection of the invention.
The invention provides following any one or more material of (a)-(c) to prepare the product for Diagnosing Cardiac disease
In application：
(a)miR-21-5p；
(b)miR-30a-3p；
(c)miR-30a-5p。
MiR-21-5p, miR-30a-3p or the miR-30a-5p provided in the present invention can be used as heart failure correlation disease
The risk of disease is estimated and detected.Discovery that can be early suffers from heart failure the risk of disease, is easy to the prevention and treatment of early stage.
In one preferred embodiment, miR-21-5p nucleotides sequence is classified as：
GGACCUUAGCUUAUCAGACUGAUG (SEQ ID No.1), miR-30a-3p nucleotides sequence is classified as
GGACCCUUUCAGUCGGAUGUUUG (SEQ ID No.2), miR-30a-5p nucleotides sequence is classified as
GGACCUGUAAACAUCCUCGACUG(SEQ ID No.3)。
Wherein, the miR-21-5p that is related to can be in the present invention：With the identical sequence of sequence shown in SEQ ID NO.1
Row, the bioactive functions piece either containing sequence shown in the sequence of sequence shown in SEQ ID NO.1 or SEQ ID NO.1
Section, or the variant of sequence shown in SEQ ID NO.1.Every sequence for possessing functional nucleotide sequence shown in SEQ ID NO.1, all should
Protection scope of the present invention is interpreted as, without should only be interpreted as and the identical sequence of sequence shown in SEQ ID NO.1.
The miR-30a-3p being related in the present invention can be：With the identical sequence of sequence shown in SEQ ID NO.1, or
Person contains the sequence of sequence shown in SEQ ID NO.1, or the bioactive functions fragment of sequence shown in SEQ ID NO.1, or
The variant of sequence shown in person SEQ ID NO.1.Every sequence for possessing functional nucleotide sequence shown in SEQ ID NO.1, all it should be understood
For protection scope of the present invention, without should only be interpreted as and the identical sequence of sequence shown in SEQ ID NO.1.
The miR-30a-5p being related in the present invention can be：With the identical sequence of sequence shown in SEQ ID NO.1, or
Person contains the sequence of sequence shown in SEQ ID NO.1, or the bioactive functions fragment of sequence shown in SEQ ID NO.1, or
The variant of sequence shown in person SEQ ID NO.1.Every sequence for possessing functional nucleotide sequence shown in SEQ ID NO.1, all it should be understood
For protection scope of the present invention, without should only be interpreted as and the identical sequence of sequence shown in SEQ ID NO.1.
In one preferred embodiment, product is reagent or kit.
In one preferred embodiment, heart disease is heart failure.
Present invention also offers a kind of reagent for Diagnosing Cardiac disease, including following (A)-(C) any one or it is more
Kind material：
(A) it is used for the primer for detecting miR-21-5p；
(B) it is used for the primer for detecting miR-30a-3p；
(C) it is used for the primer for detecting miR-30a-5p.
Present invention also offers a kind of kit for Diagnosing Cardiac disease, including following (A)-(C) any one or
Many kinds of substance or above-mentioned reagent：
(A) it is used for the primer for detecting miR-21-5p；
(B) it is used for the primer for detecting miR-30a-3p；
(C) it is used for the primer for detecting miR-30a-5p.
MiR-21-5p, miR-30a-3p or the miR-30a-5p provided in this kit can be used as heart failure related
The risk of disease is estimated and detected.Discovery that can be early suffers from heart failure the risk of disease, is easy to the prevention and treatment of early stage.
In one preferred embodiment, the sense primer for detecting miR-21-5p is miR-21-5p-F, downstream
Primer is miR-21-5p-R；Sense primer for detecting miR-30a-3p is miR-30a-3p-F, anti-sense primer miR-
30a-3p-R；Sense primer for detecting miR-30a-5p is miR-30a-5p-F, anti-sense primer miR-30a-5p-R.On
The nucleotide sequence for stating primer is as shown in the table：
| Primer | Sequence (5 ' -3 ') | Sequence number |
| miR-21-5p-F | GGACCTTAGCTTATCAGACTGATG | SEQ ID No.4 |
| miR-30a-3p-F | GGACCCTTTCAGTCGGATGTTTG | SEQ ID No.5 |
| miR-30a-5p-F | GGACCTGTAAACATCCTCGACTG | SEQ ID No.6 |
The equal source TaKaRa kits Mir-X MIran of miR-21-5p-R, miR-30a-3p-R and miR-30a-5p-R
First-Strand Synthesis Kit Cat#638313。
In one preferred embodiment, heart disease is heart failure.
In addition, present invention also offers a kind of detection method of any one or more material of following (a)-(c), including：
Extract sample rna and carry out fluorescent quantitation qPCR reactions, obtain the relative expression of any one or more material of (a)-(c)
Amount；
Wherein (a)-(c) is：
(a)miR-21-5p；
(b)miR-30a-3p；
(c)miR-30a-5p。
MiR-21-5p, miR-30a-3p or the miR-30a-5p provided in the present invention can be used as heart failure correlation disease
The risk of disease is estimated and detected.Discovery that can be early suffers from heart failure the risk of disease, is easy to the prevention and treatment of early stage.
In one preferred embodiment, the detection method of any one or more material of following (a)-(c), including：
Step (a)：The collection of clinical sample and case exclusion standard；
Step (b)：The processing of clinical sample, including blood plasma separation, the collection of miRNA, qualitative and quantitative analysis；
Step (c)：Quantitative fluorescent PCR reacts；
Step (d)：The statistical analysis of data.
In one preferred embodiment, sample rna is extracted using Trizol methods.
Present invention also offers above-mentioned miR-21-5p, miR-30a-3p or miR-30a-5p detection method, by means of reality
When quantitative PCR technology come count Microrna change, have analysis sample requirement it is small, automaticity is high, avoid pollution etc.
Advantage.
In order to contribute to it is clearer understand present disclosure, be described in detail as follows in conjunction with specific embodiment.
The collection of the clinical sample of embodiment 1 and case exclusion standard
The present embodiment have chosen in October, 2016 in May, 2017 in Hospital Attached to Medical College, Qingdao Univ.'s angiocarpy disease altogether
The sample of disease, all cases obtain informed consent from patient or family members, and relevant patient information is complete and is established by regulation
Clinical data database.Exclusion standard includes：(1) adverse cardiac patient such as ST-Elevation Acute heart infarction patient is merged, sternly
Weight valvular cardiac, purpose avoid influence of the heart organic disease to research itself；(2) acute infection or slow is merged
Property inflammation is in acute attack stage, the influence for avoiding inflammatory stimulus from falling ill heart failure in itself；(3) malignant tumour or tight is merged
Weight systemic disease patient, such as kidney failure, hepatic failure patients；(4) doubtful drug abuse and alcoholic patients；(5) refusal participates in this experiment
Patient.
The experimental specimen of embodiment 2
The blood sample of all patients is acquired in art in strict accordance with collection of specimens specification.Extracted in patient's blood vessel
10mL peripheral bloods stand 30min, 4 DEG C of 500g centrifugations with the heparin tube of common EDTA anti-freezings, being immediately placed on 4 DEG C of refrigerators
20min, purpose remove the blood cell composition remained in blood plasma, draw upper plasma, are transferred in a new centrifuge tube, continue
4 DEG C of 1500g centrifuge 20min, it is intended to thoroughly remove the material such as remaining cell fragment in blood plasma, gained blood plasma is dispensed, with -80
Refrigerator is spent to preserve.
The extraction of the blood plasma total serum IgE of embodiment 3
The extraction of blood plasma total serum IgE takes 500 μ patient's L blood plasma marks respectively using the Trizol reagents of Invitrogen companies
This, is divided into 250 μ L mono- and manages, totally two pipes.
It is separated
(1) 750 μ L Trizol reagents are separately added into, are turned upside down, fully mixes, is stored at room temperature 5min；
(2) often pipe presses 1:5 ratio adds 200 μ L chloroform, turns upside down, and thoroughly mixes, until becoming milky white completely
Untill color, 10min is stored at room temperature；
(3) 10min is centrifuged under the conditions of 4 DEG C of 12000g；
(4) it is careful to draw the μ L of upper strata colourless aqueous phase 600 high-visible three layers in pipe after centrifuging, now pay attention in not sucking
Interbed, put quality before quantity.
Precipitate RNA
(5) 600 isometric μ L isopropanols are added, while 3 μ L of addition glycogen, mixing of turning upside down, -20 DEG C of refrigerators are quiet
Put overnight；
(6) 12000g centrifuges 10min under the conditions of 4 DEG C, abandons supernatant, pays attention to not outwelling the RNA of the ttom of pipe of precipitation.
Wash RNA
(7) the alcohol 1mL, 12000g for adding 75% DEPC water configuration centrifuge 10min under the conditions of 4 DEG C, abandon supernatant；
(8) absolute ethyl alcohol 1mL, 12000g are added and centrifuge 10min under the conditions of 4 DEG C, abandoned supernatant, be stored at room temperature 5-10min,
Treat that RNA is spontaneously dried.
Dissolve RNA
(9) diluted and dissolved with 15 μ L RNA free water, it is standby.
The fluorescent quantitation qPCR of embodiment 4 reacts
(1) RNA concentration and purity testing：Apply NaNodrop instruments and carry out RNA concentration and purity testing.Strictly press
The working specification that photograph is closed is used, and is returned to zero by the use of DEPC water as blank control, takes 1 μ L RNA samples to be detected,
Obtain the sample rna concentration.General A260/A280 numerical value can use between 1.8 and 2.1.
(2) cDNA synthesis：The explanation for adding A kits in strict accordance with TaKaRa one-step method is operated, and concrete component is such as
Under：
| Reagent | Usage amount (μ L) |
| mRQ buffer(2×) | 5 |
| RNA sample | 2 |
| mRQ Enzyme mix | 1.25 |
| ddH2O | 1.75 |
| Total volume | 10 |
Reverse transcription reaction, specific procedure are as follows：
| Temperature (DEG C) | Time (min) |
| 37 | 60 |
| 85 | 5 |
| 4 | 5 |
Brief centrifugation, 90 μ L sterilized water constant volume is added to 100 μ L systems.
(3) pcr amplification reaction, the preparation of reaction system and response procedures are set
Reaction condition and result treatment：
3 repetition reacting holes are set, and point sample is expanded into 96 orifice plate PCR reaction plates by three-step approach：
The specificity of pcr amplification reaction is analyzed according to solubility curve, obtains the CT values of each sample.By the use of U6 as internal reference,
U6 sense primer is U6-F：5’-CTCGCTTCGGCAGCACATATACT-3’(SEQ ID No.7)；U6 anti-sense primer is
U6-R：5 '-ACGCTTCACGAATTTGCGTGTC-3 ' (SEQ ID No.8), according to 2-ΔΔCTMethod calculates gene relative quantification
Expression.
The statistical analysis of embodiment 5
Statistical analysis is existed using the statistical softwares of GraphPad Prism 5 or SPSSwin softwares or medcalc softwares
Windows systems support lower progress statistical analysis.
As a result such as Figure 1A, Figure 1B, Fig. 1 C, Fig. 2A, Fig. 2 B, Fig. 2 C, Fig. 3 A, Fig. 3 B, Fig. 3 C, Fig. 4 A, Fig. 4 B and Fig. 4 C institutes
Show.Wherein, Figure 1A is to detect inspections of the miR-21-5p in heart failure patient and check sample using fluorescence quantifying PCR method
Survey；Figure 1B is to detect detections of the miR-30a-3p in heart failure patient and check sample using fluorescence quantifying PCR method；Figure
1C is to detect detections of the miR-30a-5p in heart failure patient and check sample using fluorescence quantifying PCR method；From result
In it can be seen that heart failure sample be decreased obviously with control group than result, illustrate selected by three targets can be used for the Predict analysis heart
Decline sample.Fig. 2A is that the ROC of the testing result in heart failure patient and check sample to detection miR-21-5p is analyzed；Figure
2B is that the ROC of the testing result in heart failure patient and check sample to detection miR-30a-3p is analyzed；Fig. 2 C are to inspection
The ROC for surveying testing results of the miR-30a-5p in heart failure patient and check sample is analyzed；It can be seen that from ROC results
Related three targets of Confirmation Of Number can be used for Predict analysis heart failure sample.Fig. 3 A are to detecting miR-21-5p and miR-
Correlation analysis of the 30a-3p in sample is detected；Fig. 3 B are to detecting miR-21-5p and miR-30a-5p in sample is detected
Correlation analysis；Fig. 3 C are the correlation analysis in sample is detected to detection miR-30a-3p and miR-30a-5p；From right
Than can be seen that between target that there is correlation two-by-two in result.Fig. 4 A are to detecting miR-21-5p and clinical examination NT-
Correlation analysis of the proBNP in sample is detected；Fig. 4 B are that detection miR-21-5p and clinical examination NT-proBNP is being detected
Correlation analysis in sample；Fig. 4 C are related in sample is detected to clinical examination NT-proBNP to detection miR-21-5p
Property analysis；It can be seen that target has correlation two-by-two with clinical criteria NT-proBNP from comparing result.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
The present invention is described in detail with reference to foregoing embodiments for pipe, it will be understood by those within the art that：Its according to
The technical scheme described in foregoing embodiments can so be modified, either which part or all technical characteristic are entered
Row equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention technology
The scope of scheme.
Sequence table
<110>University Of Qingdao
<120>MiRNA application, product and detection method using it
<160> 8
<170> SIPOSequenceListing 1.0
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<213>Artificial sequence ()
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acgcttcacg aatttgcgtg tc 22
Claims (10)
1. application of following any one or more material of (a)-(c) in the product for Diagnosing Cardiac disease is prepared：
(a)miR-21-5p；
(b)miR-30a-3p；
(c)miR-30a-5p。
2. application according to claim 1, it is characterised in that the nucleotide sequence of the miR-21-5p such as SEQ ID
Shown in No.1, the nucleotide sequence of the miR-30a-3p is as shown in SEQ ID No.2, the nucleotides sequence of the miR-30a-5p
Row are as shown in SEQ ID No.3.
3. application according to claim 1, it is characterised in that the product is reagent or kit.
4. according to the application described in claim any one of 1-3, it is characterised in that the heart disease is heart failure.
A kind of 5. reagent for Diagnosing Cardiac disease, it is characterised in that the reagent include following (A)-(C) any one or
Many kinds of substance：
(A) it is used for the primer for detecting miR-21-5p；
(B) it is used for the primer for detecting miR-30a-3p；
(C) it is used for the primer for detecting miR-30a-5p.
6. a kind of kit for Diagnosing Cardiac disease, it is characterised in that it is any one that the kit includes following (A)-(C)
Reagent described in kind or many kinds of substance or claim 5：
(A) it is used for the primer for detecting miR-21-5p；
(B) it is used for the primer for detecting miR-30a-3p；
(C) it is used for the primer for detecting miR-30a-5p.
7. the kit described in reagent according to claim 5 or claim 6, it is characterised in that
The primer for being used to detect miR-21-5p has the sense primer of sequence and such as SEQ ID as shown in SEQ ID No.4
The anti-sense primer of sequence shown in No.5；
The primer for being used to detect miR-30a-3p has the sense primer of sequence and such as SEQ ID as shown in SEQ ID No.6
The anti-sense primer of sequence shown in No.7；
The primer for being used to detect miR-30a-5p has the sense primer of sequence and such as SEQ ID as shown in SEQ ID No.8
The anti-sense primer of sequence shown in No.9.
8. the kit described in reagent according to claim 5 or claim 6, it is characterised in that the heart disease
For heart failure.
9. a kind of detection method of any one or more material of following (a)-(c), it is characterised in that detect and state method bag
Include：Extract sample rna and carry out fluorescent quantitation qPCR reactions, obtain the relative table of any one or more material of (a)-(c)
Up to amount；
(a)-(c) be：
(a)miR-21-5p；
(b)miR-30a-3p；
(c)miR-30a-5p。
10. detection method according to claim 9, it is characterised in that extract sample rna using Trizol methods.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711105641.3A CN107858419A (en) | 2017-11-10 | 2017-11-10 | MiRNA application, product and detection method using it |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711105641.3A CN107858419A (en) | 2017-11-10 | 2017-11-10 | MiRNA application, product and detection method using it |
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