CN107012199A - A kind of method that miRNA is detected in blood plasma and serum - Google Patents
A kind of method that miRNA is detected in blood plasma and serum Download PDFInfo
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- CN107012199A CN107012199A CN201610061103.8A CN201610061103A CN107012199A CN 107012199 A CN107012199 A CN 107012199A CN 201610061103 A CN201610061103 A CN 201610061103A CN 107012199 A CN107012199 A CN 107012199A
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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
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- 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
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention discloses a kind of method that miRNA is detected in blood plasma and serum, specifically, blood plasma and blood serum sample are directly carried out heating and the processing of pre-treatment buffer by this method without RNA extraction step, compared with traditional detection method, the detection method is not only convenient, quick, low cost, with higher accuracy, higher sensitivity, higher specific, higher detection efficiency, in addition, it has also been found that may be used as detecting the miRNA combination thing of the biomarker of mammary gland carcinogenesis and transfer in blood plasma.
Description
Technical field
The present invention relates to biological technical field, in particular it relates to which a kind of detect miRNA's in blood plasma and serum
Method.
Background technology
Mitchell PS in 2008 etc. have found that miRNA can exist in the form of stable in blood plasma, it is to avoid endogenous
The degraded of RNase.The same year Shlomit G etc. confirms that miRNA stable in blood plasma and other body fluid can be deposited
, and express spectra changes with different pathological physiological status.Because peripheric venous blood obtains simple and patient
It is acceptant, overcome the difficulties such as sample size deficiency so that applications of the circulation miRNA in medical diagnosis on disease is ground
Study carefully the focus as research.
The most frequently used and effective miRNA detection of expression technologies are fluorescence quantitative PCR methods.Current general step
For：Sample rna is extracted, is reversed as miRNA cDNA, fluorogenic quantitative detection miRNA expressions.The
One step extracting sample rna is more difficult.Because blood plasma miRNA content is relatively low, if extracted when sample size is less
The deficiency follow-up test of RNA amounts.The most frequently used plasma circulation RNA extraction process continues to use traditional TriZol extraction process：
TriZol is cracked, chloroform extraction, isopropanol precipitating, the washing of 75% ethanol, the dissolving of DEPC water.The method is carried
Take efficiency low, it is big to sample size requirements, and also the residual of organic reagent can suppress follow-up PCR reactions, cause
MiRNA detection efficiencies decline.The kit extraction process of in the market is：Cracking, extraction, magnetic bead or adsorption column
Absorption, is washed, dissolving.This method would generally use magnetic bead or adsorption column, can cause losing, breaking for RNA
Bad RNA integrality is big to sample requirement amount and costly.
Also, traditional blood plasma miRNA detection method is complicated, time-consuming, cost is high, and repeatability is low, no
Beneficial to clinical expansion and application.
Therefore, this area is in the urgent need to developing a kind of simplification detection method, reducing cost, improve repeatability
The method that miRNA is detected in blood plasma and serum.
The content of the invention
It is an object of the invention to provide a kind of simplification detection method, reduce cost, improve repeatability in blood
The method that miRNA is detected in slurry and serum.
First aspect present invention provide a kind of nondiagnostic exempt from extracting to test sample carry out miRNA inspections
The method of survey, methods described includes step：
(i) test sample is provided, the test sample is blood plasma and blood serum sample, by the test sample and in advance
Buffer solution mixing is handled, the first mixture is formed, wherein the pre-treatment buffer includes non-ionic surfactant
Agent；
(ii) first mixture is heated, obtains the second mixture through processing；With
(iii) second mixture is directly used as template, detects the species of the miRNA in the test sample
And/or content.
In another preference, in step (i), the pre-treatment buffer is selected from down including one or more
The nonionic surface active agent of group：Polysorbas20 (TWEEN-20), tween 21 (TWEEN-21), tween
40 (TWEEN-40), polysorbate60 (TWEEN-60), Tween61 (TWEEN-61), Tween 80 (TWEEN-80),
Sorbimacrogol oleate100 (TWEEN-81), polysorbate85 (TWEEN-85), Triton X-100, NP-40 or its combination.
In another preference, the nonionic surface active agent includes polysorbas20 (TWEEN-20).
In another preference, the concentration (V/V) of the nonionic surface active agent is 0.5-10%, it is preferred that
1-5%, more preferably, 2-4%.
In another preference, solvent for use is water.
In another preference, the concentration of the nonionic surface active agent is 0.5-10wt%, it is preferred that
1-5wt%, more preferably, 2-4wt%.
In another preference, in step (i), the pH of the pre-treatment buffer is 6-9, it is preferred that 7-9,
More preferably, 7-8.
In another preference, the pre-treatment buffer also includes Tris-HCl buffer solutions.
In another preference, the concentration of the Tris-HCl buffer solutions is 25-150mM, it is preferred that
30-100mM, more preferably, 30-80mM.
In another preference, the pre-treatment buffer also includes metal-chelator.
In another preference, the metal-chelator is selected from the group：EDTA, EGTA or its combination.
In another preference, the metal-chelator includes EDTA.
In another preference, the concentration of the metal-chelator is 0.5-2.0mM, it is preferred that 0.5-1.5mM,
More preferably, 0.8-1.2mM.
In another preference, in step (ii), the heating-up temperature is 60-80 DEG C, it is preferred that 70-75
℃。
In another preference, in step (iii), detected with the method for quantitative fluorescent PCR in test sample
MiRNA content.
In another preference, the miRNA comes from mammal, preferably people.
In another preference, the step of methods described does not include extracting RNA.
In another preference, methods described is non-treatment nondiagnostic.
In another preference, methods described, which is detected in the test sample, is selected from the group the one or more of (A)
MiRNA species and/or quantity：miR-20b、miR-106a、miR-16.
In another preference, methods described, which is detected in the test sample, is selected from the group the one or more of (B)
MiRNA species and/or quantity：miR-671、miR-151、miR-433、miR-409.
Second aspect of the present invention provide a kind of miRNA combination thing, the composition include following miRNA or by
Following miRNA is constituted：miR-20b、miR-106a、miR-16、miR-671、miR-151、miR-433、
And miR-409.
In another preference, the composition includes at least one or whole miRNA for being selected from the group (A)：
miR-20b、miR-106a、miR-16。
In another preference, the composition includes at least one or whole miRNA for being selected from the group (B)：
miR-671、miR-151、miR-433、miR-409。
Third aspect present invention provides a kind of purposes of composition described in second aspect of the present invention, for preparing inspection
Survey the diagnostic reagent or kit of Metastasis in Breast Cancer.
In another preference, the composition is also act as detecting the life of mammary gland carcinogenesis and transfer in blood plasma
Thing mark.
In another preference, the kit includes：(i) miRNA combination described in second aspect of the present invention
Thing is used as reference substance；(ii) label or specification, wherein, the label or specification indicate the kit
For detection or Diagnosis of Breast metastasis of cancer.
In another preference, the diagnostic reagent is monoclonal antibody.
In another preference, described detection Metastasis in Breast Cancer is blood plasma detection.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and (such as implementation below
Example) in specifically describe each technical characteristic between can be combined with each other, so as to constitute new or preferred skill
Art scheme.As space is limited, no longer tire out one by one herein and state.
Brief description of the drawings
Fig. 1 shows the comparison of the method and conventional method of the present invention.
Fig. 2 shows the preferred of the best approach.Wherein, 2A：The comparison of three kinds of different disposal Sample Methods；
2B：Three kinds of methods detect miR-16, display M3 (chemical modification and heat denatured are combined) inspection respectively
Survey efficiency highest.
Fig. 3 shows the accuracy of the detection method.The plasma sample of different volumes is taken, quantitative detection
MiR-16, its content and sample volume proportional, wherein, 3A：Using M3 (chemical modification and
Heat denatured is combined), quantitatively detect blood plasma miR-16；3B：MiR-16 amplification cycles numbers；3C：Will be upper
State amplification cycles number and be scaled miRNA relative expression quantities, and sample volume linear proportional relation.
Fig. 4 shows the specificity of the detection method.The exogenous of different volumes is added in same plasma sample
Tiny RNA unisp6, unisp6 is successfully be detected using this method, and its content for detecting is with adding
Amount is consistent.MiR-16 is as internal reference, and its content does not change with exogenous unisp6 addition.Wherein,
4A：Quantitatively detect the abundance of unisp6 in blood plasma；4B：Detection and miR-16 unrelated unisp6.
Fig. 5 compares the detection of traditional detection method and this programme application by detecting miR-92a and let-7b
Method.Wherein, 5A shows that blood plasma direct measuring method (P) detection efficiency extracts detection better than traditional RNA
Method (R)；5B shows that two methods detect differences of the let-7b in different samples respectively, and result is consistent
's；5C shows that two methods detect differences of the miR-92a in different samples respectively, and result is consistent.
Fig. 6 show miR-106a and miR-671 in blood plasma can as Metastasis in Breast Cancer biomarker.
Wherein, 6A shows 4 normal persons and 6 patient with breast cancers (including 3 non-diverting patients of cancer cell and 3 cancers
Cell shifts patient) blood plasma miRNA examination, find the miRNA of 7 unconventionality expressions；6B passes through more various
This checking, it was demonstrated that miR-106a conspicuousnesses in metastatic breast cancer blood plasma are raised；6C passes through more multisample
Checking, it was demonstrated that miR-671 in metastatic breast cancer blood plasma conspicuousness lower.
The sample that Fig. 7 displays are heated, using blood plasma direct measuring method, is able to detect that miR-16, and
The sample of SDS processing, fails to detect miR-16 using blood plasma direct measuring method.
The sample that Fig. 8 displays are heated, using blood plasma direct measuring method, is able to detect that miR-21, and
The sample of SDS processing, fails to detect miR-21 using blood plasma direct measuring method.
The sample that Fig. 9 displays are heated, using blood plasma direct measuring method, is able to detect that 5s rRNA, and
The sample of SDS processing, fails to detect 5s rRNA using blood plasma direct measuring method.
Embodiment
The present inventor develops a kind of new detection miRNA side first by in-depth study extensively
Method, blood plasma and blood serum sample are carried out this method into heating and nonionic surfactant is handled, and resulting is mixed
Compound extracts without RNA and is directly used as template, the content for detecting miRNA in blood plasma and serum.The inspection
The characteristics of testing result of survey method has high accuracy, high sensitivity, high specific, high detection efficiency.
In addition, the present inventors have additionally discovered that a series of miRNA molecules, can as Diagnosis of Breast metastasis of cancer label.
On this basis, inventor completes the present invention.
As used herein, term " pre-treatment buffer ", " denaturation buffer " are used interchangeably, and are referred both to
One kind contains polysorbas20 (0.5-10%V/V), EDTA (0.8-1.2mM) and Tris-HCl (30-80mM)
Solution.
Detection method
The invention provides a kind of new method that can be used for detecting Microrna (miRNA).
Detection method is employed to be heated and denaturation buffer (including non-ionic table to blood plasma and serum
Face activating agent) coprocessing, resulting mixture is directly used in miRNA detection without RNA extraction steps.
The comparison of the method and conventional method of the present invention is as shown in Figure 1.In the present invention, one it is typical
MiRNA detection methods include following basic step：
(i) test sample is provided, the test sample is blood plasma and blood serum sample, by the test sample and in advance
Buffer solution mixing is handled, the first mixture is formed, wherein the pre-treatment buffer includes non-ionic surfactant
Agent；
(ii) first mixture is heated, obtains the second mixture through processing；With
(iii) second mixture is directly used as template, detects the species of the miRNA in the test sample
And/or content.
In the present invention, a kind of the step of preferred detection method includes：
(1) denaturation buffer is prepared：2.5% (v/v) Tween 20,50mmol/L Tris-HCl, 1
mmol/L EDTA
(2) 5uL blood plasma adds the isometric denaturation buffers of 5uL, reacts 10 minutes, 75 DEG C of water-bath 5min enter
Ice cooling, centrifugation.
(3) conventional commercial miRNA Reverse Transcriptase kits are utilized, are cDNA, dilution by miRNA reverse transcriptions
10-100 times standby.
(4) quantitative fluorescent PCR reacts, and expands miRNA, gene expression abundances of the detection miRNA in plasma sample.
16uL reaction systems：Primer (2.5pmol/uL) before 1uL cDNA, 1uL miRNA to be measured, 1uL is general
Primer (2.5pmol/uL), 8uL SYBR fluorometric reagents, 5uL H afterwards2O。
Reaction condition：50℃、2min；95℃、10min；40 circulations：95 DEG C, 15s, 60 DEG C, 1min.
The detection method of the present invention not only eliminates RNA extraction step, also with testing result high accuracy,
The characteristics of high specific, high sensitivity, high detection efficiency.
Detect the biomarker of mammary gland carcinogenesis and transfer
Present invention also offers miRNA combination thing, it is used as detecting mammary gland carcinogenesis and transfer in blood plasma
Biomarker.
In the present invention, miRNA combination thing of the invention is selected from the group：miR-20b、miR-106a、miR-16、
MiR-671, miR-151, miR-433, miR-409 or its combination.
Wherein, miR-20b, miR-106a and miR-16 can be special in the blood plasma of metastatic breast cancer patient
Different in nature high expression；MiR-671, miR-151, miR-433 and miR-409 are metastatic breast cancer patient's
Being capable of specific low expression in blood plasma.
Advantages of the present invention mainly includes：
(1) detection method eliminates RNA extraction step, and method is simple, and cost is lower.
(2) testing result of the detection method has high accuracy, high specific, the spy of high detection efficiency
Point.
(3) the detection method sensitivity is higher, it is to avoid the miRNA losses in blood plasma RNA preparation process.
(4) detection method is fast facilitated, and is compared with conventional method, and detection time shortens half.
(5) detection method avoids, using organic solvents such as Trizol, eliminating conventional method remaining organic
Influence of the solvent to subsequent detection.
(6) above-mentioned advantage is based on, the detection method is more suitable for clinical examination and detection.
(7) find first, miRNA combination thing of the invention is used as breast cancer in detection blood plasma and sent out
Raw and transfer biomarker.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only used for
The bright present invention rather than limitation the scope of the present invention.The experiment side of unreceipted actual conditions in the following example
Method, generally according to normal condition, such as Sambrook et al., molecular cloning：Laboratory manual (New York:
Cold Spring Harbor Laboratory Press, 1989) described in condition, or according to manufactory
Condition proposed by business.Unless otherwise indicated, otherwise percentage and number are weight or volume percentage and again
Measure number.
1. method
(1) denaturation buffer is prepared：2.5% (v/v) Tween 20,50mmol/L Tris-HCl, 1
mmol/L EDTA
(2) 5uL blood plasma adds the isometric denaturation buffers of 5uL, reacts 10 minutes, 75 DEG C of water-bath 5min enter
Ice cooling, centrifugation.
(3) conventional commercial miRNA Reverse Transcriptase kits are utilized, are cDNA, dilution by miRNA reverse transcriptions
10-100 times standby.
(4) quantitative fluorescent PCR reacts, and expands miRNA, gene expression abundances of the detection miRNA in plasma sample.
16uL reaction systems：Primer (2.5pmol/uL) before 1uL cDNA, 1uL miRNA to be measured, 1uL is general
Primer (2.5pmol/uL), 8uL SYBR fluorometric reagents, 5uL H2O afterwards.
Reaction condition：50℃、2min；95℃、10min；40 circulations：95 DEG C, 15second, 60
℃、1min。
2. the method for optimization
Three kinds of different albuminous degeneration methods are compared first, and M1 is heat denatured, and M2 is denaturation buffer
Denaturation is learned, M3 is that chemical modification and heat denatured are combined, and compares distinct methods to miRNA in blood plasma
In release and the influence that quantitatively detects.
Using above-mentioned three kinds of different methods, the expression that quantitatively have detected miR-16 in same plasma sample is rich
Degree, as a result as shown in Figure 2 A and 2B.As a result show, M3 detection efficiencies highest (period is minimum), M1 times
It, M2 efficiency is minimum (period is maximum).
As a result show, it is feasible using plasma protein denaturation directly detection miRNA methods, and (the change of M3 methods
Learn denaturation and heat denatured combination) for blood plasma miRNA detection efficiency highest.
3. the checking of method accuracy
Take 1 respectively, 4,8ul blood plasma, using M3 methods describeds, blood plasma miR-16 is quantitatively detected, to verify
The difference of miR-16 amounts in different volumes blood plasma.
As a result as shown in Fig. 3 A, 3B, 3C.As a result show, 1,4,8ul blood plasma distinguish corresponding miR-16
Amplification cycles number is 31.8,29.6,28.5.Plasma volumes are bigger, and period is smaller, meet expection.Will
Above-mentioned amplification cycles number is scaled miRNA relative expression quantities, and sample volume linear proportional relation, R2Reach
0.999, as a result show, the detection of detection method of the invention to blood plasma miRNA has high accuracy.
4. the specific checking of method
In a plasma sample, a kind of exogenous RNA (unisp6) is added.This RNA sheets in blood plasma
Body is not present.Set and give 0 in three groups of experiments, equivalent blood plasma respectively, 1,4ul unisp6, recycle
M3 methods describeds, quantitatively detect the abundance of unisp6 in blood plasma, to verify this method to some specific miRNA
The specificity of detection.As a result as shown in Figure 4 A.As a result show, unisp6 is detected not in 0ul experimental group
Arrive；And be able to detect that in 1ul and 4ul experimental group, and its abundance relative ratio ≈ 4, and unisp6
Addition is flux matched, and as a result demonstrate this method has high specific to RNA quantitative analyses.
In above-mentioned system, detection and miR-16 unrelated unisp6, as a result as shown in Figure 4 B.As a result show,
MiR-16 gene expression abundance is consistent in three groups of systems, and further demonstrate this method has to RNA quantitative analyses
High specific.
5. the checking of detection efficiency
One group of experiment, relatively more traditional extraction RNA method and the method for the present invention are designed, for same blood
Sample miRNA detections are starched, by the different extension rates of cDNA, are taken equivalent to two kinds from 0.02ul blood plasma
The cDNA of method, detects miRNA let-7b and miR-92a respectively.As a result as shown in Figure 5A.As a result show,
Compared with traditional analysis method, method of the invention has a higher detection efficiency, and Δ Ct values differ 0.7~
1.1, doubled equivalent to detection efficiency or so.
Another set experiment, relatively more traditional extraction RNA method and the method for the present invention are designed, for detecting
Certain miRNA different expression in two kinds of different blood plasma.Two methods of inventor's, have detected sample respectively
Let-7b and miR-92a expression in S4 and S5.As a result as illustrated in figs.5 b and 5 c.As a result show, two kinds of sides
Method detects sample S4 and S5 let-7b, Δ Ct ≈ 3, and the let-7b equivalent to S4 is higher than S5 8 times, and
And two methods detect sample S4 and S5 mIR-92a, Δ Ct ≈ 2, the let-7b equivalent to S4 compares S5
It is high 4 times.The detection efficiency that the experiment further demonstrates the method for the present invention is very high.
6. miRNA is detected in serum
Peripheral blood is collected to not plus in the heparin tube of anti-coagulants, is stored at room temperature 30 minutes, 1500rpm centrifuges 5 points
Clock, collects supernatant, obtains test serum sample.
(1 and 2) are detected to the miRNA (such as miR-16) in serum sample respectively in aforementioned manners.
As a result show, M3 methods (chemical modification and heat denatured combination) can successfully be detected serum miRNA,
But for same blood sample, blood plasma miRNA content is higher than serum miRNA contents (C1：C2 ＞ 130%),
Part miRNA in serum preparation process may be attributed to lose with associated proteins coprecipitation.
As a result show, can directly detect the miRNA of serum, and M3 methods (chemical modification and heat denatured
With reference to) for blood plasma miRNA detection efficiency highest.
7. clinical practice
Before by miR-96 gene chip technology, six plasma of breast cancer patients samples and 4 years are analyzed
Age matching healthy women plasma sample, wherein patient three be non-diverting breast cancer, another three be occur turn
The breast cancer of shifting, have found three miRNA in the special high expression of metastatic breast cancer patients blood plasma
(miR-20b, miR-106a, miR-16), and four in the special low expression of metastatic breast cancer patients blood plasma
MiRNA (miR-671, miR-151, miR-433, miR-409).As shown in Fig. 6 A, 6B and 6C.
Using the blood plasma direct measuring method M3 (chemical modification and heat denatured are combined) of the present invention, receive again
Collected three non-diverting plasma of breast cancer patients, three shift plasma of breast cancer patients, three health
The blood plasma of women, is verified respectively to above-mentioned miRNA, it is found that miR-106a suffers from metastatic breast cancer
The special high expression of person's blood plasma, miR-671 is in the special low expression of metastatic breast cancer patients blood plasma.The two knots
Fruit it is consistent with genechip detection result before, illustrate pass through different samples, the analysis of distinct methods, really
Accepting the two miRNA can be as metastatic breast cancer patients blood plasma's biomarker, with clinical practice
Value and potentiality, the early diagnosis available for Metastasis in Breast Cancer.
Comparative example 1
Method is with 1, and difference is, substitutes denaturation buffer with SDS, 5ul blood plasma is taken, respectively with heating
Reason or isometric 10%SDS processing, it is small to three then using the miRNA direct Detection Methods of the present invention
RNA (such as miR-16, miR-21,5srRNA) detected, as a result as Figure 7-9.
As a result as Figure 7-9.As a result show, the sample of heat denatured processing, amplification is good, utilizes blood
Direct measuring method is starched, miR-16, miR-21 and 5s rRNA is able to detect that, and the sample of SDS denaturation treatments
This, without amplification curve, fails to detect above-mentioned miRNA using blood plasma direct measuring method.
As a result show, with SDS processing, it is impossible to three Micrornas (such as miR-16, miR-21,5s rRNA)
Detected.
Comparative example 2
Method is with 1, and difference is, denaturation buffer is substituted with SDS, heating is carried out to blood plasma and isometric
10%SDS processing, then using the present invention miRNA direct Detection Methods, to three Micrornas (such as miR-16,
MiR-21,5s rRNA) detected, as a result show, handled simultaneously with heating and SDS, do not expand song
Line.
As a result show, blood plasma is heated and SDS processing simultaneously, it is impossible to three Micrornas (such as
MiR-16, miR-21,5s rRNA) detected.
All documents referred in the present invention are all incorporated as reference in this application, just as each document
It is individually recited as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read,
Those skilled in the art can make various changes or modifications to the present invention, and these equivalent form of values equally fall within this Shen
Please appended claims limited range.
Claims (10)
1. a kind of method that miRNA detections are carried out to test sample for exempting from extracting of nondiagnostic, it is characterised in that
Methods described includes step：
(i) test sample is provided, the test sample is blood plasma and blood serum sample, by the test sample and in advance
Buffer solution mixing is handled, the first mixture is formed, wherein the pre-treatment buffer includes non-ionic surfactant
Agent；
(ii) first mixture is heated, obtains the second mixture through processing；With
(iii) second mixture is directly used as template, detects the species of the miRNA in the test sample
And/or content.
2. the method as described in claim 1, it is characterised in that in step (i), the pretreatment buffering
Liquid includes one or more nonionic surface active agent being selected from the group：Polysorbas20 (TWEEN-20), tween
21 (TWEEN-21), polysorbate40 (TWEEN-40), polysorbate60 (TWEEN-60), Tween61 (TWEEN-61),
Tween 80 (TWEEN-80), sorbimacrogol oleate100 (TWEEN-81), polysorbate85 (TWEEN-85), Triton X-100,
NP-40 or its combination.
3. the method as described in claim 1, it is characterised in that in step (i), the pretreatment buffering
The pH of liquid is 6-9, it is preferred that 7-9, more preferably, 7-8.
4. the method as described in claim 1, it is characterised in that the pre-treatment buffer also includes Tris-HCl
Buffer solution.
5. the method as described in claim 1, it is characterised in that the pre-treatment buffer also includes metal chelating
Mixture.
6. method as claimed in claim 5, it is characterised in that the metal-chelator is selected from the group：EDTA、
EGTA or its combination.
7. the method as described in claim 1, it is characterised in that in step (ii), the heating-up temperature is
60-80 DEG C, it is preferred that 70-75 DEG C.
8. the method as described in claim 1, it is characterised in that in step (iii), use quantitative fluorescent PCR
Method detection test sample in miRNA content.
9. a kind of miRNA combination thing, it is characterised in that the composition includes following miRNA or by as follows
MiRNA is constituted：MiR-20b, miR-106a, miR-16, miR-671, miR-151, miR-433 and miR-409.
10. the purposes of composition as claimed in claim 9, it is characterised in that turn for preparing detection breast cancer
The diagnostic reagent or kit of shifting.
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