CN103160507A - MiRNA serum marker capable of detecting liver cirrhosis and application thereof - Google Patents
MiRNA serum marker capable of detecting liver cirrhosis and application thereof Download PDFInfo
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Abstract
The invention relates a microRNA marker which is used for distinguishing liver cirrhosis and normal tissues, in particular to the micro RNA marker has-miR-181b which is used for distinguishing the liver cirrhosis and the normal tissues. According to certification of inspection, the specified microRNA marker can effectively distinguish the liver cirrhosis and the normal tissues. The invention further relates to the application of the microRNA has-miR-181b marker and a reagent kit which is used for detecting the liver cirrhosis.
Description
Technical field
The present invention relates to biological technical field, more specifically, the present invention relates to can be used for distinguishing the microRNA mark hsa-miR-181b of liver cirrhosis sample and normal sample.Through testing identity, this specific microRNA mark can be distinguished liver cirrhosis sample and normal sample very effectively.The invention still further relates to the test kit for detection of liver cirrhosis.
Background technology
Liver cirrhosis (liver cirrhosis) is a kind of common chronic hepatopathy of China, can cause liver damage by one or more reasons, and liver is carrying out property, diffusivity, fibrous lesions.Be embodied in liver cell diffusivity degeneration necrosis, the regeneration of proliferation of fibrous tissue and liver cell nodositas appears then, these three kinds of changes are staggered carrying out repeatedly, and liver lobule structure and blood circulation approach are reconstructed gradually as a result, makes liver distortion, hardening and cause liver cirrhosis.The early stage non-evident sympton of this disease, a series of portal hypertension and hepatic insufficiencyes in various degree appear in stage, until it is dead the complication such as upper gastrointestinal hemorrhage, hepatogenic encephalopathy to occur.Therefore, the diagnosis as early as possible of liver cirrhosis just more seems urgent.
The genesis of anything all is under the internal and external reasons acting in conjunction, and is interior because main, outer because auxiliary.Gene is as the hereditary medium of life, organism sick, old,, be in the internal cause status on basis in dead.Overwhelming majority genes are by transcribing the product nucleus ribosomal ribonucleic acid, then translation generates protein and brings into play biological function.
MicroRNA (miR or miRNA, Microrna) is that a class extensively exists in more high most eukaryotes, and length is the single stranded RNA molecule of 18-26 base approximately.It can combine with target site on some mRNA specifically by basepairing rule, causes that said target mrna degraded or translation suppress, and then at post-transcriptional level, target gene is regulated and controled.
MicroRNA derives from the approximately initial transcription product of long-chain RNA (Pri-miRNA) of 1000bp of length, and the Pri-miRNA molecule is sheared through the Drosha enzyme in nucleus and formed the approximately miRNA precursor (Pre-miRNA) with loop-stem structure of 60-80nt of length.After Pre-miRNA is transported to kytoplasm, further cut into the double-stranded miRNA that is about 22nt by the Dicer enzyme.After double-stranded miRNA untied, ripe miRNA entered the reticent mixture (RNA-induced silencing complex, RISC) of RNA induced gene, with complementary mRNA fully or incomplete pairing, degraded said target mrna or check its expression.
Although microRNA shared proportion in cell total rna is very little, but because it can produce regulating and controlling effect to all mRNA with target site efficiently, microRNA still can't neglect in the growth of organism and even generation, the evolution role of disease.
Yet, up to now, this area is understood very few for the microRNA relevant to hepatic diseases (especially liver cirrhosis), so this area is in the urgent need to separation flags thing microRNA further, especially with generation, transfer, the recurrence of liver cirrhosis or detect relevant microRNA.
Summary of the invention
Purpose of the present invention just be to provide a kind of new, can be used for microRNA mark of distinguishing by liver cirrhosis sample and normal sample and uses thereof.
Another object of the present invention is to provide chip and the test kit that detects described disease.
In a first aspect of the present invention, a kind of miRNA of separation is provided, described miRNA is selected from:
(i) miRNA of sequence as shown in SEQ ID NO:1, or
(ii) with the miRNA of sequence complementation shown in SEQ ID NO:1.
In another preference, described miRNA separates from the people.
In a second aspect of the present invention, precursor miRNA a kind of separation or artificial constructed is provided, miRNA as described in first aspect present invention can be sheared and be expressed as to described precursor miRNA in people's cell.
In a third aspect of the present invention, a kind of polynucleotide of separation are provided, described polynucleotide can be become precursor miRNA by people's cell transcription, and described precursor miRNA can be sheared and be expressed as miRNA as described in first aspect present invention in people's cell.
In another preference, described polynucleotide have the structure shown in formula I:
Seq
Forward-X-Seq
Oppositely
Formula I
In formula I,
Seq
ForwardFor becoming at people's cells the nucleotide sequence of described miRNA;
Seq
OppositelyFor with Seq
ForwardBasically the nucleotide sequence of complementation or complete complementary;
X is for being positioned at Seq
ForwardAnd Seq
OppositelyBetween intervening sequence, and described intervening sequence and Seq
ForwardAnd Seq
OppositelyNot complementary;
And the structure shown in formula I forms the secondary structure shown in formula II after changing people's cell over to:
In formula II, Seq
Forward, Seq
OppositelyWith stating as defined above of X,
‖ is illustrated in Seq
ForwardAnd Seq
OppositelyBetween the base complementrity pair relationhip that forms.
In a fourth aspect of the present invention, a kind of carrier is provided, described carrier contains the described miRNA of first aspect present invention, or the described polynucleotide of this aspect third aspect.
In a fifth aspect of the present invention, the purposes of the described miRNA of first aspect present invention is provided, described miRNA is for the preparation of chip or the test kit of distinguishing liver cirrhosis sample and normal sample.
In a sixth aspect of the present invention, a kind of miRNA chip is provided, described miRNA chip comprises:
Solid phase carrier; And
Be fixed in order the oligonucleotide probe on described solid phase carrier, described oligonucleotide probe is specifically corresponding to the sequence shown in SEQ ID NO:1.
In another preference, described oligonucleotide probe contains:
Complementary land; And/or
The joining region that is connected with solid phase carrier.
In a seventh aspect of the present invention, the purposes of the described miRNA chip of sixth aspect present invention is provided, described miRNA chip is for the preparation of the test kit of distinguishing liver cirrhosis sample and normal sample.
In a eighth aspect of the present invention, a kind of test kit is provided, contain the described miRNA chip of sixth aspect present invention in described test kit.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can making up mutually between specifically described each technical characterictic in below (eg embodiment), thus consist of new or preferred technical scheme.As space is limited, this tired stating no longer one by one.
Description of drawings
Fig. 1 has shown relative expression's level of the hsa-miR-181b of liver cirrhosis sample and normal sample.
Embodiment
The inventor by detecting the microRNA express spectra level of liver cirrhosis sample serum and normal sample serum, therefrom filters out specific microRNA:hsa-miR-181b through research for a long time and widely first.Through testing identity, hsa-miR-181b can distinguish cirrhotic tissue sample and normal sample very effectively as the microRNA mark.Completed on this basis the present invention.
Particularly, inventor's real-time PCR system, the TaqMan probe method detects miR-181b, the expression of U6.Adopt Δ Ct method (Ct
MiR-181b-Ct
U6) miR-181b differential expression in liver cirrhosis and normal population serum relatively.The measurable sample of this microRNA is that its prediction accuracy reaches 80% from liver cirrhosis sample or normal sample.Based on microRNA of the present invention, can be developed to test kit, be used for distinguishing liver cirrhosis sample and normal sample.
MiRNA and precursor thereof
The invention provides the new miRNA that finds of a class from the people.As used herein, described " miRNA " refers to a kind of RNA molecule, from forming the transcript processing of miRNA precursor.Ripe miRNA has 18-26 Nucleotide (nt) (more particularly approximately 19-22nt) usually, does not also get rid of the miRNA molecule with other number Nucleotide.MiRNA can be detected by the Northern trace usually.
The miRNA in people source can be separated from people's cell.As used herein, " separation " refers to that material separates (if natural substance, primal environment is namely natural surroundings) from its primal environment.There is no separation and purification as the polynucleotide under the native state in active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, for separation and purification.
MiRNA can be from precursor miRNA (Precursor miRNA, Pre-miRNA) processing, and described precursor miRNA can be folded into a kind of stable stem ring (hair clip) structure, and described loop-stem structure length is generally between 50-100bp.Described precursor miRNA can be folded into stable loop-stem structure, and the stem both sides of loop-stem structure comprise basically complementary two sequences.Described precursor miRNA can be natural or synthetic.
Precursor miRNA can be sheared and generate miRNA, and described miRNA can be basically complementary with at least a portion sequence of the mRNA of encoding gene.As used herein, " basically complementary " refers to that the sequence of Nucleotide is enough complementary, can interact in a kind of foreseeable mode, as forming secondary structure (as loop-stem structure).It is complementary that the nucleotide sequence of two usually, " basically complementary " has 70% Nucleotide between mutually at least; Preferably, it is complementary having 80% Nucleotide at least; Preferred, it is complementary having 90% Nucleotide at least; Further preferred, it is complementary having 95% Nucleotide at least; As 98%, 99% or 100%.Usually, two enough can have maximum 40 unmatched Nucleotide between complementary molecule; Preferably, have maximum 30 unmatched Nucleotide; Preferred, have maximum 20 unmatched Nucleotide; Further preferred, have maximum 10 unmatched Nucleotide, as have 1,2,3,4,5,8,11 unmatched Nucleotide.
As used herein, " stem ring " structure also is known as " hair clip " structure, refer to a kind of nucleic acid molecule, it can form a kind of secondary structure that comprises double-stranded region (stem), described double-stranded region is formed by two zones (being positioned at on a part) of this nucleic acid molecule, the both sides of two double-stranded parts of regional apportion; It also comprises at least one " ring " structure, comprises non-complementary nucleic acid molecule, i.e. the strand zone.Even two zones of this nucleic acid molecule are not complete complementaries, the double-stranded part of Nucleotide also can keep double-stranded state.For example, insertion, disappearance, replacement etc. can cause not complementary or this zonule self formation loop-stem structure of a zonule or the secondary structure of other form, yet these two zones still can be basically complementary, and interact in foreseeable mode, form the double-stranded region of loop-stem structure.Loop-stem structure is well-known to those skilled in the art, and after the nucleic acid that has obtained a nucleotide sequence with primary structure, those skilled in the art can determine whether this nucleic acid can form loop-stem structure usually.
MiRNA of the present invention has the sequence as shown in SEQ ID NO:1.In order to improve stability or other character of miRNA, also can add at least one end of described miRNA at least one protectiveness base, as " TT " etc.
Antisense oligonucleotide
According to miRNA sequence provided by the present invention, can design its antisense oligonucleotide, described antisense oligonucleotide can be lowered the expression of corresponding miRNA in vivo.As used herein, " antisense oligonucleotide (antisense-oligonucleotides; AS-Ons or ASO) " is called again " antisense nucleotide ", refers to that length is about DNA molecular or RNA molecule or its analogue of 18-26nt (more particularly approximately 19-22nt).
In the present invention, described " antisense oligonucleotide " also comprises the modified antisense nucleotide that adopts as based on means acquisitions such as nucleic acid lock or nucleic acid chains backbone modification technology, described modification does not change the activity of antisense oligonucleotide substantially, more preferably, described modification can improve stability, activity or the result for the treatment of of antisense oligonucleotide.Nucleic acid lock (locked nucleic acid, LNA) typically refers to the modification technique that 2 ' Sauerstoffatom and the 4 ' carbon atom of ribose is coupled together by a methylene bridge.LNA can extend the serum half-life of miRNA, improves the target affinity, reduces scope and the degree of the effect of missing the target.The antisense drug that technical development goes out based on the nucleic acid chains modification of framework is in solubility, and the aspects such as nuclease-resistant degraded are improved greatly, and is easy to a large amount of synthetic.The backbone modification method of oligonucleotide has multiple, comprises the sulfo-method, is for example sulfo-deoxynucleotide chain with deoxynucleotide chain thio-modification.The method is that the Sauerstoffatom of the phosphate bond on the DNA skeleton is alternative with sulphur atom, can resist nuclease degradation.Should be understood that any can keep the most of of described antisense oligonucleotide or all active modifications be included in the present invention.
As optimal way of the present invention, antisense oligonucleotide is carried out the nucleic acid lock modify; More preferably also carry out thio-modification.
After transferring to antisense oligonucleotide of the present invention in human body, they can obviously lower the expression of relevant miRNA.
The polynucleotide construction
According to miRNA sequence provided by the present invention, can design the polynucleotide construction of the miRNA that can be processed to affect corresponding mrna expression after being imported into, be also the amount that described polynucleotide construction can raise corresponding miRNA in vivo.Therefore, the invention provides a kind of polynucleotide (construction) of separation, described polynucleotide (construction) can be become precursor miRNA by people's cell transcription, and described precursor miRNA can and be expressed as described miRNA by people's cell shearing.
As a kind of optimal way of the present invention, described polynucleotide construction contains the structure shown in formula I:
Seq
Forward-X-Seq
Oppositely
Formula I
In formula I,
Seq
ForwardFor becoming at cells the nucleotide sequence of described miRNA, Seq
OppositelyFor with Seq
ForwardBasically complementary nucleotide sequence; Perhaps, Seq
OppositelyFor becoming at cells the nucleotide sequence of described miRNA, Seq
ForwardFor with Seq
ForwardBasically complementary nucleotide sequence;
X is for being positioned at Seq
ForwardAnd Seq
OppositelyBetween intervening sequence, and described intervening sequence and Seq
ForwardAnd Seq
OppositelyNot complementary;
Structure shown in formula I forms the secondary structure shown in formula II after changing cell over to:
In formula II, Seq
Forward, Seq
OppositelyWith stating as defined above of X;
‖ is illustrated in Seq
ForwardAnd Seq
OppositelyBetween the base complementrity pair relationhip that forms.
Usually, described polynucleotide construction is positioned on expression vector.Therefore, the present invention also comprises a kind of carrier, and it contains described miRNA, or described polynucleotide construction.Described expression vector also contains promotor, replication orgin and/or marker gene etc. usually.Method well-known to those having ordinary skill in the art can be used for building expression vector required for the present invention.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described expression vector preferably comprises one or more selected markers, with the phenotypic character of the host cell that is provided for selecting transforming, as kalamycin, gentamicin, Totomycin, amicillin resistance.
Chip
The microRNA chip of expression spectrum contains a nearly hundreds of probe usually, contains multiple microRNA, the principle of utilizing DNA double chain homologous complementary content of contained various microRNA in the detection sample on full genomic level.Therefore, can be at one time the transcriptional level of the microRNA in full genome range in sample to be tested be detected.
Utilize miRNA sequence of the present invention, can also prepare corresponding miRNA chip, and then study the regulative mode of its express spectra and miRNAs.
On the other hand, the present invention also provides a kind of chip for analyzing the miRNA express spectra, and described chip can be used for distinguishing liver cirrhosis sample and normal sample.
Described miRNA chip of the present invention comprises solid phase carrier and is fixed in order oligonucleotide probe on described solid phase carrier, and described oligonucleotide probe comprises the sequence shown in SEQ ID NO:1.
Particularly, can design suitable probe according to miRNA of the present invention, be fixed on solid phase carrier, form " oligonucleotide arrays ".Described " oligonucleotide arrays " refer to have the addressable point array of (namely with distinctive, addressable address is the position of feature), a coupled characteristic oligonucleotide is all contained in each addressable point.As required, oligonucleotide arrays can be divided into a plurality of inferior battle arrays.
Described solid phase carrier can adopt the various common used materials in gene chip field, such as but not limited to nylon membrane, and the slide of the slide of modifying through active group (as aldehyde radical, amino etc.) or silicon chip, unmodified, plastic sheet etc.
The preparation of described miRNA chip can be adopted the conventional manufacture method of biochip known in the art.For example, if what solid phase carrier adopted is to modify slide or silicon chip, 5 ' end of probe contains amido modified poly-dT string, oligonucleotide probe can be mixed with solution, then adopt point sample instrument that its point is being modified on slide or silicon chip, be arranged in predetermined sequence or array, then spending the night by placement fixes, and just can obtain miRNA chip of the present invention.If it is amido modified that nucleic acid does not contain, its preparation method also can reference: Wang Shenwu chief editor's " gene diagnosis technology-on-radiation operational manual "; J.L.erisi, V.R.Iyer, P.O.BROWN.Exploring the metabolic and genetic control of gene expression on a genomic scale.Science, 1997; 278:680 and Ma Li people, the Jiang Zhonghua chief editor. biochip. Beijing: Chemical Industry Press, 2000,1-130.
On the other hand, the present invention also provides a kind of method by miRNA express spectra in miRNA chip detection people tissue, comprises step:
(1) provide the RNA sample that separates from people's tissue, on described RNA, marker is set;
(2) RNA with (1) contacts with described chip, makes the oligonucleotide probe generation hybridization on described RNA and solid phase carrier, thereby forms " oligonucleotide probe-RNA " binary complex on solid phase carrier;
(3) detect the marker of the binary complex of (2) formation, thereby determine the express spectra of corresponding miRNA in people's tissue.
The method of extracting RNA from people's tissue is method well known to those skilled in the art, comprises the Trizol method.
Preferred, in step (1), isolate the RNA sample from people's tissue tissue after, the RNA sample is suitably processed, have the RNA of certain-length with enrichment, described length is between 10-100 (small fragment RNA) generally.Through after above-mentioned processing, utilize these small fragment RNAs to carry out follow-up hybridization, can improve the accuracy that chip is caught miRNA like this.Those skilled in the art can isolate the RNA with certain fragment length easily, such as adopting gel electrophoresis to separate.
It is also method well known to those skilled in the art that RNA is carried out mark, and it can realize by add the method with the marker of RNA specific binding in when hybridization, and described marker is such as being labelling groups.Described labelling groups includes but not limited to: digoxin molecule (DIG), biotin molecule (Bio), fluorescein and derivative biomolecules (FITC etc.) thereof, other fluorescence molecule (as Cy3, Cy5 etc.), alkaline phosphatase (AP), horseradish peroxidase (HRP) etc.These marks and marking method thereof have been all routine techniques well-known in the art.
When above-mentioned RNA and miRNA chip are hybridized, can first miRNA chip and prehybridization damping fluid be carried out prehybridization.
Solid-phase hybridization between RNA of the present invention and miRNA chip carries out according to the classical way of this area, and the general personnel in this area easily determine relevant damping fluid, probe and the optimum condition of concentration of specimens, prehybridization temperature, hybridization temperature and time etc. according to experience.Perhaps also can be with reference to described in " molecular cloning experiment guide ".
Then the acquisition of informations such as the position on the miRNA chip, intensity are treated measurement information according to marking signal.If amplified production fluorophor mark also can directly obtain and treat measurement information with fluorescence detection device (as laser confocal scanning instrument Scanarray 3000 etc.).
Detection kit
The present invention also provides a kind of test kit, contains chip of the present invention in described test kit.Described test kit can be used for detecting the express spectra of miRNA; Or be used for distinguishing liver cirrhosis sample and normal sample, and preferred, also contain the marker that is useful on the labeled rna sample in described test kit, and the substrate corresponding with described marker.
In addition, also can comprise in described test kit for extracting the required all ingredients such as RNA, PCR, hybridization, colour developing, include but not limited to: extract, amplification liquid, hybridization solution, enzyme, contrast liquid, nitrite ion, washing lotion, antibody etc.
In addition, also can comprise working instructions and/or chip image analysis software in described test kit.
Major advantage of the present invention
(a) the invention provides a kind of microRNA mark that can be used for distinguishing liver cirrhosis sample and normal sample.
(b) can very effectively distinguish liver cirrhosis sample and normal sample by microRNA mark of the present invention.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Embodiment 1
The sample source
Collect 30 routine liver cirrhosis and 15 routine normal population serum, these samples come from the Xinhua Hospital Digestive System Department.Above-mentioned all samples obtain all agreement by the Ethics Committee of WHO cooperative association of Shanghai City government authorization.The clinical data of tissue samples comprises: sex, age, pathological grading etc.
Embodiment 2
The blood serum designated object screening
MiRNA extracting: with the mirVana of Ambion company
TMIsolation kit extracting, with reference to the test kit specification sheets operation of the said firm, concrete operations are as follows: get 600 μ l serum, add the miRNA lysate of 500 μ l, put upside down mixing, place 10min on ice.Add isopyknic sour phenol-chloroform mixed solution, the spiral mixing, the centrifugal 5min of 10000rpm room temperature gets the upper strata water.Add the dehydrated alcohol of 1/3rd volumes in above-mentioned Organic extraction thing.Be added to after mixing and collect in pillar.The centrifugal 15s of 10,000rpm speed.Collect flowing liquid, add the dehydrated alcohol of 2/3rds volumes, mixing.Mixture is added on a new pillar, and the centrifugal 15s of 10,000rpm speed discards effluent liquid.700 μ l washing lotions 1 are added on pillar centrifugal 15s.500 μ l washing lotions 2/3 are added to respectively on pillar, and centrifugal 15s repeats once.Pillar is put in a new collection tube.95 ℃ of preheatings are without RNA enzyme water, and 50 μ l preheatings are added to elution samples on pillar without RNA enzyme water, the centrifugal 1min of 10,000rpm speed.Repeat once.
Electrophoresis check: with denaturing formaldehyde sepharose and urea-denatured polyacrylamide gel electrophoresis, the integrity of Ethidum Eremide staining examine RNA.Concentration with UV spectrophotometer measuring RNA.
MiRNA reverse transcription: the RNA sample TaqMan of Applied Biosystems company
MicroRNA reverse transcription test kit uses the special primer with neck ring to carry out reverse transcription, ripe miRNAs only detected during with assurance real time PCR.Each reverse transcription reaction 5ng RNA, target gene and U6 carry out reverse transcription in same reaction system.The reverse transcription reaction condition is: 16 ℃, and 30min; 42 ℃, 30min; 85 ℃, 5min.
PCR in real time amplification: with the synthetic MGB probe in detecting PCR product of ABI company, carry out relative quantitative assay with U43 as internal reference.Relative quantification real-time PCR reaction is reacted with ABI 7300 systems.Each sample repeats three holes.Following reaction system is at 95 ℃ of denaturation 5min, 95 ℃ of sex change 30s afterwards, 60 ℃ of 1min, 40 circulations.Adopt Luo Shi (Roche) LightCycler
480 real-time PCR systems detect miR-181b, the expression of U6 with the TaqMan probe method.Adopt Δ Ct method (Ct
MiR-181b-Ct
U6) miR-181b differential expression in liver cirrhosis and normal population serum relatively.
Compare with the normal people, miR-181b expresses in serum of cirrhosis patients obviously and raises (P＜0.05), sees Fig. 1.
The sequence of hsa-miR-181 is as shown in SEQ ID NO:1, in human genomic sequence, there are 2 precursor sequence: be positioned at No. 1 karyomit(e) miR-181b-1 (MIR:MI0000270) and be positioned at No. 9 chromosomal mir-181b-2 (MIR:MI0000683).
Embodiment 3
Preparation miRNA chip
Convert miRNA sequence provided by the invention (SEQ ID NO:1) to complementary sequence, add the catenation sequence of 10-20nt according to features such as the GC that produces sequence compare at sequence two ends; Core sequence is different, and catenation sequence is also different.Catenation sequence can be produced at random by program, and the probe that catenation sequence and core sequence form meets the following conditions:
1) in probe sequence, the quantity of same Nucleotide (A, C, G, T) can not surpass 50% of sequence sum;
2) any continuous A, T or the quantity of C, G can not surpass 25% of sequence sum;
3) G, C content account for the 40%-60% of sequence sum;
4) probe sequence can not be from hybridization, and namely in probe sequence, the length of complementary fragment can not surpass 30% of probe length.
For making stable being combined on slide of synthetic probe, adopt conventional method to carry out glycosyl modified at 5 ' end of synthetic rear probe.
The point system of chip: first the surface of slide is carried out alkylation and modify, to improve binding ability.Adopt conventional chip point sample method to carry out point sample, in order to detect the repeatability of cross experiment, each probe is 3-6 hybridization point of point on slide.
Embodiment 4
The test kit preparation
The Chip Packaging of preparation in embodiment 3 is good, be placed in a box together with working instructions, consist of test kit.
The detection validation of chip
To obtain a plurality of liver cirrhosis samples (comprising 30 routine liver cirrhosis samples and the normal sample of 30 examples) from hospital, press method preparation and the mark microRNA of embodiment 1-2, press the chip of the method preparation of embodiment 3, detect with double-blind method.According to the existence of microRNA mark shown in the present whether and the upper downward situation that is in harmonious proportion come judgement sample.Wherein, positive control and negative control are respectively known liver cirrhosis sample and normal sample.
Result shows, comprises the chip of species specificity microRNA of the present invention, and its exactness is 90%, can effectively distinguish liver cirrhosis sample and normal sample.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. the miRNA of a separation, described miRNA is selected from:
(i) miRNA of sequence as shown in SEQ ID NO:1, or
(ii) with the miRNA of sequence complementation shown in SEQ ID NO:1.
2. miRNA as claimed in claim 1, is characterized in that, described miRNA separates from the people.
3. a separation or artificial constructed precursor miRNA, miRNA as claimed in claim 1 or 2 can be sheared and be expressed as to described precursor miRNA in people's cell.
4. the polynucleotide of a separation, described polynucleotide can be become precursor miRNA by people's cell transcription, and described precursor miRNA can be sheared and be expressed as miRNA as claimed in claim 1 or 2 in people's cell.
5. polynucleotide as claimed in claim 4, is characterized in that, described polynucleotide have the structure shown in formula I:
Seq
Forward-X-Seq
Oppositely
Formula I
In formula I,
Seq
ForwardFor becoming at people's cells the nucleotide sequence of described miRNA;
Seq
OppositelyFor with Seq
ForwardBasically the nucleotide sequence of complementation or complete complementary;
X is for being positioned at Seq
ForwardAnd Seq
OppositelyBetween intervening sequence, and described intervening sequence and Seq
ForwardAnd Seq
OppositelyNot complementary;
And the structure shown in formula I forms the secondary structure shown in formula II after changing people's cell over to:
In formula II, Seq
Forward, Seq
OppositelyWith stating as defined above of X,
‖ is illustrated in Seq
ForwardAnd Seq
OppositelyBetween the base complementrity pair relationhip that forms.
6. a carrier, is characterized in that, described carrier contains the described miRNA of claim 1 or 2, or the described polynucleotide of claim 4 or 5.
7. the purposes of the described miRNA of claim 1, is characterized in that, for the preparation of chip or the test kit of distinguishing liver cirrhosis sample and normal sample.
8. miRNA chip, described miRNA chip comprises:
Solid phase carrier; And
Be fixed in order the oligonucleotide probe on described solid phase carrier, described oligonucleotide probe is specifically corresponding to the sequence shown in SEQ ID NO:1.
9. the purposes of the described miRNA chip of claim 8, is characterized in that, for the preparation of the test kit of distinguishing liver cirrhosis sample and normal sample.
10. a test kit, contain miRNA chip claimed in claim 8 in described test kit.
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| WO2011072177A2 (en) * | 2009-12-09 | 2011-06-16 | Aviir, Inc. | Biomarker assay for diagnosis and classification of cardiovascular disease |
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| WO2011072177A2 (en) * | 2009-12-09 | 2011-06-16 | Aviir, Inc. | Biomarker assay for diagnosis and classification of cardiovascular disease |
Non-Patent Citations (2)
| Title |
|---|
| BAOCAN WANG ET AL: "miR-181b Promotes hepatic stellate cells proliferation by targeting p27and is elevated in the serum of cirrhosis patients", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
| 李莉 等: "MicroRNAs与病毒性肝炎的研究进展", 《热带医学杂志》 * |
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