CN105803082A - miR-572 serving as marker for differential diagnosis of postoperation restenosis of lower extremity atherosclerotic occlusive disease and reagent kit thereof - Google Patents

Abstract

The invention provides application of miR-572 serving as a marker for differential diagnosis of postoperation restenosis of lower extremity atherosclerotic occlusive disease and a reagent kit for the differential diagnosis of postoperation restenosis of lower extremity atherosclerotic occlusive disease through the miR-572. By means of the miR-572, the diagnosis which is high in flexibility, strong in specificity, capable of reflecting recovery condition of disease, convenient and easy to use and low in cost is achieved. The miR-572 has good clinical application prospect and wide commercial suppliers.

Description

MiR-572 is as arterial obliterans of lower extremity postoperative restenosis Differential Diagnosis labelling Thing and test kit thereof

Technical field

The invention belongs to field of biological technology detection, be specifically related to a kind of arterial obliterans of lower extremity postoperative restenosis mirror Other diagnostic marker and test kit thereof.

Background technology

Limb chronic ischemia comes from the lower limb blood supply insufficiency that lower limb atherosclerosis is narrow or obturation causes, and is most common The people that makes lose class disease, referred to as an arterial obliterans of lower extremity of normal walking ability¹, mainly show as intermittent lame Row, lower limb rest pain and gangrene etc., have a strong impact on the quality of life even threat to life of patient².Along with China's living standard Improvement and aged tendency of population, the sickness rate of arterial obliterans of lower extremity is also improving year by year.

Developing rapidly recently as medical skill, the treatment of arterial obliterans of lower extremity also presents diversification, e.g., Coronary artery bypass grafting, Endarterectomy art and interventional therapy etc. all can have efficient recovery blood supply, improve symptom³.In clinic diagnosis, due to During arterial obliterans of lower extremity patients's disease, often the age is higher, and it is many often to merge coronary heart disease, heart failure, cerebral infarction etc. Planting disease, comprehensive assessment patient's ordinary circumstance is poor to the toleration of coronary artery bypass grafting and Endarterectomy art.Interventional therapy is with its wound The advantages such as little, post-operative recovery is fast relative to other two kinds of Therapeutic Method in the operative treatment of arterial obliterans of lower extremity more by Extensively application⁴.Meanwhile, there is focus of attention to " temporarily irrigating raising " phenomenon^5,6,7, i.e. arterial obliterans of lower extremity causes Limb chronic ischemia at PTCA or and STENTS, inaccessible section arterial perfusion is temporarily improved, for reparation and the obturation of ischemic tissue Certain time has been striven in the formation of section periarterial collateral circulation, but when getting involved the tremulous pulse after intervening and being the narrowest or inaccessible, Original pathological changes will be made to be in subclinical ischemic state.Therefore, postoperative restenosis becomes affects some patients late result One of key issue.

At present, for detection methods such as the CTA of postoperative restenosis, angiographys, because diagnosis and treatment resource-constrained, inspection fee are held high Your many factors such as grade, tends not in patient's long term follow-up conventional enforcement, and needs patient intermittent claudication, quiet again occur Just being carried out during breath pain even gangrene, now generally requiring performs the operation again loses the chance of limb salvage treatment, severe patient the most Jeopardize patient vitals⁷.Therefore, the early diagnosis biomarker finding arterial obliterans of lower extremity postoperative restenosis becomes One of key scientific problems urgently to be resolved hurrily, this is to taking effective remedy measures, monitoring PD in time and being avoided serious Complication occurs significant.

Ranasinghe etc.⁸Propose the general characteristic that preferable early diagnosis index should possess: highly sensitive, specificity By force, can reflect the course of disease of disease lapse to, quick, easy-to-use, cheap, there is good potential applicability in clinical practice and business widely Industry supplier.Research for arterial obliterans of lower extremity postoperative restenosis biomarker provides theoretical standard.

During clinic diagnosis, many lab testing projects with it rapidly, can complete and to disease at bedside There is the features such as higher sensitivity and specificity, be widely applied.The great discovery Microrna of science in recent years (mircoRNA, miRNA) is that a class is widely present and the little molecule non-coding RNA of high conservative human and animal, and it is at the heart Vascular tissue's cell development is formed in exception procedure with differentiation and structure and plays particularly important role.

Recently research shows, miRNA is because having the characteristic of tissue specificity and high conservative so that it is have as disease raw The powerful potential quality of substance markers thing.Ai etc.⁹Compared with matched group by patients of acute myocardial infarction, miR-1 in combination model mice Process LAN, it was demonstrated that in patients of acute myocardial infarction circulating, miR-1 level significantly increases, and returns to foundation level after two weeks Variation tendency；Professor Jing Qing etc.¹⁰Report patients of acute myocardial infarction compared with the miR-significantly increased in Healthy People circulating 208a, its blood plasma level changes with Prognosis, and the characteristic that in peripheral blood, miR-208a occurs early than cardiac troponin, Prompting miR-208a is probably the early stage biomarker that acute myocardial infarction one is more sensitive.

Having research display, arterial obliterans of lower extremity postoperative restenosis is the healing reaction after arterial injury¹¹, it occurs After development is the Artery injury of a series of vaso-active substance and somatomedin mediation, endotheliocyte and smooth muscle cell are altogether Result with the repair process homeostasis participated in.MiRNA is reported in during multiple angiopathy develops and sends out in recent years Wave important function: have research display, miR-221 and miR-222 high expressed can suppress vascular smooth muscle cell proliferation, migration and Apoptosis function, and in endotheliocyte, miR-221 and miR-222 can promote its propagation, migrate and apoptosis；This completely phase Anti-effect may play a significant role in angiopathy develops¹³；Quintavalle M etc.¹⁴Report mechanicalness After blood vessel injury, the expression change of miR-21, miR-143 and miR-145 can affect vascular smooth muscle cell by shrinkage type to conjunction Molding converts, and the recovery of miR-21 and miR-145 expression can avoid the generation of vascular restenosis, prompting blood vessel injury to repair After multiple during restenosis

MiRNA may play this important function.So, during arterial obliterans of lower extremity postoperative restenosis whether Also the expression along with specific miRNA changes, and whether can there is arterial obliterans of lower extremity postoperative restenosis in these miRNA Potential source biomolecule label, the most not yet someone provides answer.

List of references:

1.Malas MB,Qazi U,Glebova N,Arhuidese I,Reifsnyder T,Black J,Perler BA,Freischlag JA.Design ofthe Revascularization With Open Bypass vs Angioplasty and Stenting of the Lower Extremity Trial(ROBUST):a randomized clinical trial.JAMA Surg.2014Dec；149(12):1289-95.

2.Huen KH,Chowdhury R,Shafii SM,Brewster LP,Arya S,Duwayri Y, Veeraswamy RK,Dodson TF,Rajani RR.Smoking cessation is the least successful outcome of risk factor modification in uninsured patients with symptomatic peripheral arterial disease.Ann Vasc Surg.2015Jan；29(1):42-9.

3.Iida O,Takahara M,Soga Y,Suzuki K,Hirano K,Kawasaki D,Shintani Y, Suematsu N,Yamaoka T,Nanto S,Uematsu M.Shared and differential factors influencing restenosis following endovascular therapy between TASC(Trans- Atlantic Inter-Society Consensus)II class A to C and D lesions in the femoropopliteal artery.JACC Cardiovasc Interv.2014Jul；7(7):792-8

4.Deloose K,Bosiers M,Callaert J,Verbist J,Vermassen F,Scheinert D, Torsello G,Peeters P.Primary stenting is nowadays the golden standard treatment for TASC II A&amp；B iliac lesions:The definitive MISAGO 1-year results.J Cardiovasc Surg(Torino).2014 Oct 21.[Epub ahead ofprint]

5.Yen HT,Hsieh MJ,Wu CC,Lee FY.Effect of systemic urokinase infusion after lower limb percutaneous transluminal angioplasty on limb salvage rate in patients with late-stage critical limb ischemia.Eur J Vasc Endovasc Surg.2014 Oct；48(4):414-22.

6.Landry GJ,Esmonde NO,Lewis JR,Azarbal AF,Liem TK,Mitchell EL,Moneta GL.Objective measurement of lower extremity function and quality of life after surgical revascularization for critical lower extremity ischemia.J Vasc Surg.2014 Jul；60(1):136-42.

7.Hawkins AT,Schaumeier MJ,SmithAD,Hevelone ND,Nguyen LL.When to call it a day:incremental risk of amputation and death after multiple revascularization.Ann Vasc Surg.2014Jan；28(1):35-47.

8.Ranasinghe AM,Bonser RS.Biomarkers in acute aortic dissection and other aortic syndromes.J Am Coll Cardiol.2010；56:1535-1541.

9.Rapezzi C,Longhi S,Graziosi M,Biagini E,Terzi F,Cooke RM,Quarta C, Sangiorgi D,Ciliberti P,Di Pasquale G,Branzi A.Risk factors for diagnostic delay in acute aortic dissection.Am J Cardiol.2008；102:1399-1406.

10.Nienaber CA,Eagle KA.Aortic dissection:New frontiers in diagnosis and management:Part i:From etiology to diagnostic strategies.Circulation.2003；108:628-635.

11.Yoo AR,Koh SH,Cho GW,Kim SH.Inhibitory effects of cilostazol on proliferation o fvascular smooth muscle cells(VSMCs)through suppression of the ERK1/2pathway.J Atheroscler Thromb.2010 Oct 27；17(10):1009-18.

12.Davis-Dusenbery BN,Wu C,Hata A.Micromanaging vascular smooth muscle cell differentiation and phenotypic modulation.Arterioscler Thromb Vasc Biol.2011 Nov；31(11):2370-7.

13.Liu X,Cheng Y,Yang J,Xu L,Zhang C.Cell-specific effects of miR- 221/222in vessels:molecular mechanism and therapeutic application.J Mol Cell Cardiol.2012 Jan；52(1):245-55.

14.Quintavalle M,Elia L,Condorelli G,Courtneidge SA.MicroRNA control of podosome formation in vascular smooth muscle cells in vivo and in vitro.J Cell Biol.2010 Apr 5；189(1):13-22.

Summary of the invention

The present invention is carried out for solving the problems referred to above, it is therefore intended that provide a kind of highly sensitive, high specificity, can The course of disease of reflection disease lapses to, quick, easy-to-use, cheap, there is good potential applicability in clinical practice and commercial offers widely The arterial obliterans of lower extremity postoperative restenosis label of business and corresponding detection kit.To achieve these goals, Present invention employs techniques below scheme:

The invention provides a kind of miR-572 and differentiate answering of label as arterial obliterans of lower extremity postoperative restenosis With, wherein, the sequence of miR-57 is as shown in SEQ ID NO.1.

Further, present invention also offers a kind of arterial obliterans of lower extremity postoperative restenosis identification reagent box, its Be characterised by having: blood plasma total RNA extraction reagent group, external source object of reference, RNA reverse transcription reagents group, RNA reverse transcriptase primer group, Real-time quantitative PCR reagent set, real-time quantitative PCR primer sets and real-time quantitative PCR probe groups.Wherein, RNA reverse transcriptase primer group bag Include miR-572 reverse transcriptase primer and external source object of reference reverse transcriptase primer；Real-time quantitative PCR primer sets includes that miR-572 is fixed in real time Amount PCR primer and external source ginseng real-time quantitative PCR primer；Real-time quantitative PCR probe groups includes miR-572 real-time quantitative PCR probe Real-time quantitative PCR probe is joined with external source.

In mentioned reagent box, external source object of reference is cel-miR-39-3p, cel-miR-39-3p sequence such as SEQ ID Shown in NO.2,2 ' the methylated modifications of O of all nucleotide of cel-miR-39-3p.

Invention effect and effect

The miR-572 that the present invention provides answering as arterial obliterans of lower extremity postoperative restenosis Differential Diagnosis label With and a kind of arterial obliterans of lower extremity postoperative restenosis differential diagnosis kit for miR-572, it is achieved that under to Highly sensitive, the high specificity of limb atherosclerotic occlusive disease postoperative restenosis, can reflect that the course of disease of disease lapses to, fast, easily With, cheap diagnosis, there is good potential applicability in clinical practice and commercial supplier widely.

Accompanying drawing explanation

Fig. 1 is miRNA core in the arterial obliterans of lower extremity restenosis after vascular intervention group of the present invention and matched group blood plasma Sheet experiment scanning figure (part)；

Fig. 2 is chip data supervision clustering analysis result figure, wherein, arterial obliterans of lower extremity postoperative restenosis group: N1、N2、N3、N11、N12、N13、N14、N15；Arterial obliterans of lower extremity postoperative long term follow-up rehabilitation clients: N4, N6, N8, N10、N17；Healthy volunteer: N5, N7, N9, N16.

Detailed description of the invention

The detailed description of the invention of the present invention is described below in conjunction with accompanying drawing.

One, the acquisition of miR572

Inventor, in the preliminary experiment of small sample, is obtaining patient and healthy volunteer's informed consent and is signing informed consent In the case of book, occur for the first time gathering patient and healthy volunteer's peripheric venous blood when lower limb ischemia symptom is gone to a doctor patient, and Experimenter is carried out diagnosis and treatment and long term follow-up.1, case and the selection of matched group:

Collect the 357 example arterial obliterans of lower extremity patients blood plasma's specimen planning to implement operative treatment, collect age phase simultaneously 106 imitative example healthy volunteer's plasma specimens.All sample standard deviations sign Informed Consent Form patient and family members (or volunteer) thereof Afterwards and obtain on the premise of meeting the scientific scrutiny such as ethics.

1.1 inclusive criterias:

A. lower extremities is inaccessible: first generation lower extremities obturation, non-operation theratment be (femoral artery,superficial pathological changes TASC classification A type, Type B, c-type patient)；

B. volunteer: the healthy volunteer being of the similar age；

C. arterial obliterans of lower extremity postoperative restenosis patient: Follow-up After occurs lower limb ischemia disease again in 6 months Shape, obtains radiological evidence through lower limb CTA or angiography；

D. arterial obliterans of lower extremity postoperative long term follow-up rehabilitation clients: Follow-up After occurs in 1 year that lower limb lack the most again Mass formed by blood stasis shape, obtains radiological evidence through lower limb CTA or angiography；

1.2. exclusion standard

All without lower extremity surgery history, without blood transfusion history, without hepatic and kidney function obstacle；It is associated with other artery of lower extremity beyond femoral artery,superficial (modus operandi difference will cause between group, organize interpolation for the arterial obliterans of lower extremity patient of pathological changes and TASC classification D type patient Different, therefore get rid of)；

1.3. the specimen packet gathered:

A. experimental group 1 (N=16): arterial obliterans of lower extremity postoperative restenosis patient；

Experimental group 2 (N=100): arterial obliterans of lower extremity postoperative restenosis patient；

Experimental group 3 (N=200): arterial obliterans of lower extremity postoperative restenosis patient；

B. blank group 1 (N=10): healthy volunteer；

Blank group 2 (N=100): healthy volunteer；

Blank group 3 (N=150): healthy volunteer；

C. case-control group 1 (N=16): arterial obliterans of lower extremity postoperative long term follow-up rehabilitation clients；

Case-control group 2 (N=100): arterial obliterans of lower extremity postoperative long term follow-up rehabilitation clients；

Case-control group 3 (N=150): arterial obliterans of lower extremity postoperative long term follow-up rehabilitation clients.

2, blood preparation and the collection of clinical data:

A. use EDTA-K2 anticoagulant tube to gather peripheric venous blood 5ml, and completed to be centrifuged in 1 hour: 820g, 4 DEG C, 15min, takes 16000g after supernatant, 4 DEG C, 10min；Make a record respectively with labelling after, move to RNAse-free cryopreservation tube, preserve In-80 DEG C of refrigerators.

B. make a collection of specimens the sex of source person, age, history of present illness, Smoking And Drinking history, complication (hypertension, diabetes, height Lipidemia etc.), clinical stages, iconography indices seen by preoperative MRA or CTA (according to TASC classification record obturation section, close Plug length, narrow section, stenosis and length, far-end blood supply situation etc.), these model parameters are become statistical data storehouse.

3, the screening of difference miRNAs and checking in plasma specimen:

The extracting of A.RNA: total with QIAGEN miRNeasy Mini kit based on Trizol method extracting plasma sample RNA。

B.RNA quality testing: measure the RNA suction at spectrophotometer 260nm, 280nm and 230nm wavelength with Nanodrop Receipts value, to calculate concentration and to assess purity；Carry out Denaturing Agarose Gel electrophoresis with formaldehyde electrophoresis reagents, detection RNA purity and Integrity.

C. the detection of miRNAs is circulated:

A. screening stage: the miRNAs after extracting is carried out with Agilent Human miRNA 8 × 60K chip V16.0 High flux screening；

B. Qualify Phase: RT-qPCR based on Taqman sonde method verifies candidate miRNAs.

D. the collection of data and analysis: extract initial data also with Agilent Feature Extraction (v10.7) Preserve, with Agilent GeneSpring, initial data is analyzed, carries out statistical calculation with SPSS16.0.

Patient's basic feature describes: Student ' s t-test and Fisher ' s exact test.

Between each group, miRNAs expression compares: ANOVA.

Specific miRNA is used for sensitivity and the specificity of arterial obliterans of lower extremity restenosis after vascular intervention early diagnosis Analyze: ROC curve and AUC.

MiRNAs compares with existing biomarker: repeated-measures ANOVA.

E. arterial obliterans of lower extremity restenosis after vascular intervention specificity and the high miRNAs of sensitivity are filtered out.

4, specific miRNA and the correlation analysis of patient clinical data:

A. the specific miRNA every clinical parameter with arterial obliterans of lower extremity restenosis after vascular intervention is analyzed (such as year Age, sex, clinical stages etc.), risk factor (such as hypertension, diabetes, hyperlipemia etc.), the course of disease lapse to and the index such as prognosis Between interrelated.

B. follow-up of patients, obtain latter 3 months of morbidity, 6 months, 1 year and the information of following up a case by regular visits to (symptom, image data) of 2 years, outer All venous blood specimen；

C. compare with arterial obliterans of lower extremity postoperative restenosis goldstandard Arteriography of lower extremity arteries, analyze specific MiRNA is in the quality of the aspect more relatively Arteriography of lower extremity arteries such as arterial obliterans of lower extremity postoperative restenosis early diagnosis and prognosis Gesture；Analyze and lapse to the aspect specific miRNA variation tendency compared with Arteriography of lower extremity arteries in the course of disease.

Fig. 1 is that in arterial obliterans of lower extremity restenosis after vascular intervention group and matched group blood plasma, miRNA array experiment is swept Tracing (part).

As it is shown in figure 1, by high-throughput techniques contrast arterial obliterans of lower extremity postoperative restenosis patient (A, n=8), Arterial obliterans of lower extremity postoperative long term follow-up rehabilitation clients (B, n=5) and miRNA in healthy volunteer (C, n=4) circulating Differential expression, through significance analysis obtain difference miRNA 61.

Fig. 2 is chip data supervision clustering analysis result figure.

Further through artificial nerve network model and combine chip forecast analysis on the basis of Fig. 1 result (Prediction analysis of microarrays, PAM), obtain in Fig. 2 is postoperative with arterial obliterans of lower extremity 15 miRNA such as the closely-related miR-572 of restenosis.In these miRNA, part has peripheral arterial tissue specificity, wherein The cell derived of different miRNA may be not quite similar.

In above-mentioned 15 miRNA, the diagnosis accuracy of miR-572 is 94.2%, specificity is 92.8%, sensitivity is 94.1%, belong to the miRNA differentiating that performance is stronger.

In sum, inventor proposes such hypothesis: after arterial obliterans of lower extremity postoperative restenosis occurs, periphery In arterial tissue, particularly inner membrance and the impaired cell of middle film discharge miRNA and enter blood circulation, peripheral arterial organizing specific MiRNA simultaneously also into peripheral blood, these miRNA do not exist in the peripheral blood of Healthy People and other diseases patient or Person's abundance is extremely low.These specific miRNA may imply that arterial obliterans of lower extremity postoperative restenosis occurs and vascular remodeling During play the cell of pivotal role or structure is in different process, therefore, one of them or several miRNA do not only have into Possibility for arterial obliterans of lower extremity postoperative restenosis early diagnosis biomarker, it is also possible to close with lower extremities The course of disease of plug disease postoperative restenosis lapses to and prognosis has close contact.

Two, the use of arterial obliterans of lower extremity postoperative restenosis identification reagent box

In following specific implementation process, the reagent used in corresponding steps is as follows: with purchased from Qiagen company MiRNeasy Mini Kit carries out RNA extraction, including 2ml centrifuge tube (uncovered), 1.5ml centrifuge tube (having lid), QIAzol Lysis Reagent, water, RWT buffer, RPE buffer and Rneasy Mini spin column without RNA/DNA enzyme Filter post；With the MicroRNA Reverse Transcription Kit (article No. purchased from Applied Biosystems company 4366597) RNA reverse transcription is carried out, including: 10 × Reverse Transcription Buffer, 100mM dNTPs (with dTTP)、20U/μL Rnase Inhibitor、50U/μL MμLtiScribeTM Reverse Transcriptase；With Universal PCR Master Mix II, no purchased from Applied Biosystems company UNG (article No. 4440048) carries out real-time quantitative PCR.Additionally, RNA reverse transcriptase primer and real-time quantitative PCR primer and probe are Purchased from the MicroRNA Assays (article No. 4427975) of Applied Biosystems company, including two parts, one Demultiplexing in reverse transcription is: 5 × RT primer (reverse transcription primer)；A part expands for real-time quantitative PCR, contains amplification Primer and specific probe, for: in Small RNA Assay (20 ×), i.e. this detailed description of the invention implement quantification PCR primer and Probe is pre-mixed.The miRNA assays of ABI company uses unified article No., and specifically certain miRNA has again it to number, and relates to And the Assays ID of the miR-572 of this project is: 001614；The Assays ID of external source object of reference cel-miR-39-3p is: 000200。

The detection object of the present invention is the plasma sample of patient.

1, the extraction of blood plasma total serum IgE

The extraction of the total serum IgE in blood plasma is according to the raw factory of miRNeasy Mini Kit (Qiagen, Hilden, Germany) The plasma specimen Total RNAs extraction standard specification that business Qiagen company provides is carried out；Specimen mixes during Total RNAs extraction Cel-miR-39-3p (sequence as shown in SEQ ID NO.2, all nucleotide 2 ' O methylate modification) is as the external source of PCR experiment Property object of reference, cel-miR-39-3p incorporation is 0.4pmol/200 μ L plasma specimen.Specific as follows:

(1) water without RNA/DNA enzyme is used to be made into 0.08pmol/ μ L solution cel-miR-39-3p analogies；

(2) take 200 μ L plasma/serum samples in 2ml centrifuge tube, add 1000 μ L QIAzol lysis Reagent (samples 5 times of product volume), whirlpool shakes up, and makes solution homogeneous without white precipitate；

(3) room temperature is placed 5 minutes；

(4) 5 μ L0.08pmol/ μ L cel-miR-39-3p solution are added (when being only used for the specimen extraction total serum IgE of PCR experiment Comprise this step)；

(5) adding 200 μ L chloroforms (for 1 times of sample volume), whirlpool shakes up, and makes solution homogeneous for pink；

(6) room temperature is placed 2-3 minute；

(7) under the conditions of 12000g4 DEG C centrifugal 15 minutes；

(8) take upper strata aqueous portion 600 μ L and be placed in the 2ml centrifuge tube having been provided that (untouchable middle level whiteness), Add 900 μ L dehydrated alcohol, lash for several times (please don't be centrifuged, precipitate may be produced, do not interfere with)；

(9) take 700 μ L sample, comprise precipitate, add in Rneasy Mini spin column (filtration post), soft Close the lid, under room temperature, 8000g is centrifuged 15s.Discard filtered solution；

(10) remaining sample repeats the 9th step；

(11) adding 700 μ LRWT buffer to Rneasy Mini spin column, 8000g, 15 DEG C of centrifugal 15s wash post, Discard filtered solution；

(12) adding 500 μ LRPE buffer to Rneasy Mini spin column, 8000g, 15 DEG C of centrifugal 15s wash post, Discard filtered solution；

(13) repeat the 12nd, take out after being centrifuged and considered post, do not touch filtered solution；

(14) it is placed in new 2ml centrifuge tube by filtering post, the most centrifugal 2 minutes (the centrifugal force ＞ of microcentrifuge 17000g)；

(15) it is placed in new 1.5ml centrifuge tube (having been provided that in test kit) by filtering post, adds 50 μ L without RNase water, soft Closeing the lid of sum, stands 1 minute, 8000g, and 15 DEG C are centrifuged 1 minute；

(16) take the solution 45 μ L of centrifugal gained in the 15th step and add filtration post (being changed without centrifuge tube)；Soft closes the lid Son, stands 1 minute, 8000g, and 15 DEG C are centrifuged 1 minute；

(17), gained solution take 1 μ L and use NP1000 to measure RNA mass and concentration, record A260/280, A260/230 and Concentration；Surplus solution is stored in-80 DEG C of refrigerators or is directly used in next experimentation (chip or qRT-PCR experiment).

2, reverse transcription reaction

2.1 prepare sample total serum IgE

The sample total serum IgE preserved at dissolving-80 DEG C under room temperature the total serum IgE mixing or extracting are directly used in this experiment.

Prepared by 2.2 reverse transcription primary response liquid

(1) each component agent in MicroRNA Reverse Transcription Kit is placed in dissolves on ice；

(2) carrying out dosing in polypropylene tube (PP pipe), this operation must be carried out on ice, and each component ratio is calculated by table 1； Recommend to increase the 10-20% of dosing expected response amount at this, the dosing that during to avoid moving liquid, reagent loss is brought is not enough；

The allocation list of table 1 reverse transcription primary response liquid

(3) want slowly during mixing, centrifugal make solution dissolve and be blended in bottom PT pipe (can not whirlpool shake up)；

(4) before carrying out reverse transcription, dosing need to be placed on ice.

2.3 prepare reverse transcription reaction

(1) 5x reverse transcription primer and RNA template is dissolved on ice；Before using, whirlpool concussion preserves the examination of reverse transcription primer Agent pipe makes its solution uniform, and carries out of short duration centrifugal；

(2) ready total serum IgE is carried out following operation:

1., in 15 μ L reaction systems, mix in the ratio adding 5 μ L total serum IgE (1-10ng) in every 7 μ L reverse transcription primary response liquid Close, become reverse transcription primary response liquid-total serum IgE mixed liquor；

2. want slowly during mixing, of short duration centrifugal make solution dissolve and be blended in bottom PT pipe (can not whirlpool shake up)；

3. every 12 μ L reverse transcription primary response liquid-total serum IgE mixed liquor is transferred to 0.2ml PT pipe or 96 orifice plates；

4. in corresponding pipe/hole, add 3 μ L reverse transcription primer, make reaction system reach 15 μ L；

5. plate tube is sealed, soft mixing, of short duration centrifugal (can not whirlpool shake up)；

The most at least hatch on ice 5 minutes, then, keep this state to carrying out reverse transcription reaction；

7. reactions steps and the condition of PCR instrument is set by table 2.

Table 2 reverse transcription reaction condition

3, real-time quantitative PCR (qPCR) reaction

3.1 prepare qPCR reaction

(1) each component is placed on ice, after careful inversion makes its mix homogeneously, puts back on ice；

(2) according to each component volume needed for 20 μ L system computing, (ABI recommends each reaction to carry out three repetitions, for preventing The consumption of reagent when moving liquid, increases total reagent and uses volume 20%), such as table 3；

The preparation of table 3 qPCR reaction system

(3) in microcentrifugal tube, each component reagent is mixed；

(4) careful centrifuge tube of being inverted is with mixed solution, then carries out of short duration centrifugal.

3.2 prepare qPCR Sptting plate

(1) the PCR reactant liquor that transferase 12 0 μ L prepares；

(2) optical adhesive film or optics cap shrouding are used, of short duration centrifugal.

3.3 carry out PCR reaction

(1) according to following parameter establishment experimental file:

1. pattern: standard

2. reaction system: 20 μ L

3. thermal circulation parameters such as table 4

(2) Sptting plate is inserted PCR instrument；

(3) reaction is started.

Table 4 qPCR thermal cycle sets

4, experimental data is read

(1) amplification curve is observed；

(2) set baseline and threshold value obtains experimental data.

The effect of embodiment and effect

Project in the present embodiment is preoperative for arterial obliterans of lower extremity, Follow-up After and restenosis occur series During the specific miRNA of circulating be object of study, carry out big in arterial obliterans of lower extremity postoperative restenosis spectrum of disease The quantitative verification of sample, sets up disease early diagnosis model and it is carried out independent sample checking and correction, and obtaining can conduct The miR-572 of arterial obliterans of lower extremity postoperative restenosis Differential Diagnosis label.MiR-572 is the most highly sensitive, special Property strong, and stable the most degradable；Meanwhile, for the arterial obliterans of lower extremity postoperative restenosis Differential Diagnosis of miR-572 Test kit is quick, easy-to-use, cheap, has good potential applicability in clinical practice and commercial supplier widely.For artery of lower extremity The hardening early warning of obliterans postoperative restenosis, state of illness monitoring and prognosis have searched out useful clinically lab testing and have referred to Mark.

Claims (3)

1.miR-572 differentiates the application of label as arterial obliterans of lower extremity postoperative restenosis,

Wherein, the sequence of described miR-572 is as shown in SEQ ID NO.1.

2. an arterial obliterans of lower extremity postoperative restenosis identification reagent box, it is characterised in that have:

Blood plasma total RNA extraction reagent group, external source object of reference, RNA reverse transcription reagents group, RNA reverse transcriptase primer group, real-time quantitative PCR reagent group, real-time quantitative PCR primer sets and real-time quantitative PCR probe groups,

Wherein, described RNA reverse transcriptase primer group includes miR-572 reverse transcriptase primer and external source object of reference reverse transcriptase primer,

Described real-time quantitative PCR primer sets includes miR-572 real-time quantitative PCR primer and external source ginseng real-time quantitative PCR primer,

Described real-time quantitative PCR probe groups includes miR-572 real-time quantitative PCR probe and external source ginseng real-time quantitative PCR probe.

Aortic Dissection identification reagent box the most according to claim 2, it is characterised in that:

Wherein, described external source object of reference is cel-miR-39-3p, described cel-miR-39-3p sequence such as SEQ ID NO.2 institute Show, 2 ' the methylated modifications of O of all nucleotide of described cel-miR-39-3p.