CN201381322Y - Fast miRNAs quantification PCR detection kit - Google Patents
Fast miRNAs quantification PCR detection kit Download PDFInfo
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- CN201381322Y CN201381322Y CN200920052584U CN200920052584U CN201381322Y CN 201381322 Y CN201381322 Y CN 201381322Y CN 200920052584 U CN200920052584 U CN 200920052584U CN 200920052584 U CN200920052584 U CN 200920052584U CN 201381322 Y CN201381322 Y CN 201381322Y
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Abstract
The utility model discloses a fast miRNAs quantification PCR detection kit, comprising an outer box body and an inner box body. Bearing holes are arranged on the inner box body, and the inner box body is arranged in the outer box body. The fast miRNAs quantification PCR detection kit is characterized by comprising a reagent used for preparing a miRNAs Poly (A) polymerase system, a reagent used for preparing a miRNAs reverse transcription reaction system and a reagent used for preparing a miRNAs qPCR reaction system, wherein the reagent used for preparing the miRNAs Poly (A) polymerase system comprises an rATP reagent, a PAP reagent and DEPC water, the reagent used for preparing the miRNAs reverse transcription reaction system comprises a Universal RT Primer reagent, a dNTP reagent, an M-MLV reagent, an RT Buffer reagent, an Rnasin Inhibitor reagent and DEPC water, and the reagent used for preparing a miRNAs qPCR reaction system comprises a Universal qPCR Primer reagent, an SYBRGreen Master Mix reagent and sterilization distilled water. Each reagent is respectively arranged in a corresponding reagent bottle, and each reagent bottle is respectively arranged in the bearing holes on the inner box body. The utility model has the advantages of wider applicability, more sensitive response, high repeatability and high accuracy.
Description
Technical field
The utility model relates to a kind of fast m iRNAs quantitative PCR detection kit.
Background technology
MiRNAs is the non-coding of a class, endogenic little RNA, about 20～23 the Nucleotide (nucleotides of the endogenous length of organism, nt), miRNAs is encoded by genomic dna, is transcribed into the primary transcribe (pri-miRNA) of a hundreds of Nucleotide (nt) in nucleus by rna plymerase ii.The pri-miRNA of this stem-ring structure is by the hairpin structure precursor pre-miRNA of RNase III family member Drosha shearing into about 70nt.Pre-miRNA is transported out nucleus subsequently, and further is processed into miRNAs by the RNase III family member Dicer of endochylema.Effect strategy and the siRNA of miRNAs is similar in the plant, promptly is incorporated into 3 ' of said target mrna-UTR or open reading frame by the accurate matching method of base, causes the said target mrna Transcriptional Silencing.But most Mammals miRNAs then are incorporated into 3 ' of said target mrna-UTR by the part base pairing, cause the specificity of said target mrna to be sheared or transcription repression.MiRNAs extensively is present in Eukaryotic growth and metabolic process to the gene regulating of post-transcriptional level, comprise apoptosis, cell proliferation, cytodifferentiation, metabolism of fat, insulin secretion, immunomodulatory, stress reaction etc., and present the spatial and temporal expression specificity.MiRNAs miRNAs gene in human genome occupies about 1% quantity, though in the different space-time etap, their expression amount difference, but the expression of multiple miRNAs in same cell makes cell be in a kind of intrinsic miRNAs environment, this environment is being controlled the miRNAs level of thousands of encoding gene, makes various proteic expression be in a suitable level.MiRNAs is non-encoding gene, and no poly (A) tail is conservative on evolving, and extensively is present in animal, plant, fungi and the viral genome.Found 695 miRNAs molecules at present in the mankind, but scientist's predict human genome may contain 1000 the special miRNAs that have an appointment, they regulate and control nearly 30% genetic expression.Recently, discover many miRNAs assignments of genes gene mapping in the gene locus of disease-related near, and variant expression in the tumour generating process, and the miRNAs that has can be used as the biomarker that early-stage cancer detects.MiRNAs content has just become in people's research to seem more and more important in the rapid detection different sources sample, but more existing detection techniques, reaction sensitivity, high duplication and high precision can't satisfy people's needs.
Summary of the invention
The purpose of this utility model provides a kind of miRNAs content in the rapid detection different sources sample that can be used to, and has that suitability is wider, a fast m iRNAs quantitative PCR detection kit of sensitiveer, the high duplication of reaction and high precision.
The purpose of this utility model is achieved through the following technical solutions.
A kind of fast m iRNAs quantitative PCR detection kit, comprise outter box, inner box, inner box is provided with bearing holes, inner box places in the outter box, it is characterized in that: be placed with the reagent bottle group that the reagent that is used to prepare miRNAs Poly (A) polysaccharase system is housed in the bearing holes on the described inner box, the reagent bottle group of the reagent that is used to prepare miRNAs reverse transcription reaction system is housed and the reagent bottle group of the reagent that is used to prepare miRNAs qPCR reaction system is housed, the reagent bottle group that the reagent that is used to prepare miRNAs Poly (A) polysaccharase system wherein is housed comprises the reagent bottle that rATP reagent is housed, the reagent bottle of poly (A) polysaccharase reagent is housed, the reagent bottle and the reagent bottle that DEPC water is housed of polyA polymerase Buffer reagent are housed, the reagent bottle group that the reagent that is used to prepare miRNAs reverse transcription reaction system is housed comprises the reagent bottle that Universal RT Primer reagent is housed, the reagent bottle of dNTP reagent is housed, the reagent bottle of M-MLV reagent is housed, the reagent bottle of RT Buffer reagent is housed, the reagent bottle of RNasinInhibitor reagent is housed and the reagent bottle of DEPC water is housed, the reagent bottle group that the reagent that is used to prepare miRNAs qPCR reaction system is housed comprises the reagent bottle that Universal qPCRPrimer reagent is housed, the reagent bottle and the reagent bottle that sterile purified water is housed of SYBR Green Master Mix reagent are housed.
The volume specification of rATP in the reagent bottle of the described rATP of being equipped with reagent is 10 μ l, the polyA polymerase volume specification that is equipped with in the reagent bottle of poly (A) polysaccharase reagent is 20 μ l, the RNasin Inhibitor volume specification that is equipped with in the reagent bottle of RNasin Inhibitor reagent is 10 μ l, the DEPC volume of water specification that is equipped with in the reagent bottle of DEPC water is 250 μ l, the M-MLV volume specification that is equipped with in the reagent bottle of M-MLV reagent is 10 μ l, the Universal qPCR Primer volume specification that is equipped with in the reagent bottle of Universal qPCR Primer reagent is 50 μ l, the Universal RT Primer volume specification that is equipped with in the reagent bottle of Universal RT Primer reagent is 20 μ l, the SYBR Green Master Mix volume specification that is equipped with in the reagent bottle of SYBR Green Master Mix reagent is 1000 μ l, the dNTP volume specification that is equipped with in the reagent bottle of dNTP reagent is 40 μ l, the aseptic distillation volume of water specification that is equipped with in the reagent bottle of sterile purified water is 1000 μ l, the RT Buffer 5 * RT Buffer volume specification that wherein is equipped with in the reagent bottle of RT Buffer reagent is 80 μ l, the polyA polymeraseBuffer 10 * polyA polymerase Buffer volume specification that is equipped with in the reagent bottle of polyA polymerase Buffer reagent is 20 μ l, and the reagent bottle that contains each reagent is the unified specification size.
The described reagent bottle group that the reagent that is used to prepare miRNAs Poly (A) polysaccharase system is housed all also comprises the reagent bottle that noncoding total RNA is housed, the OD of total RNA with the reagent bottle group that the reagent that is used to prepare miRNAs reverse transcription reaction system is housed
260/ OD
280Value is between 1.6～2.0, and the reagent bottle group that the reagent that is used to prepare miRNAs qPCR reaction system is housed also comprises the reagent bottle of the cDNA that dilution is housed and the reagent bottle of miRNA upstream primer is housed.
Described outter box passes through thin-film package.
The utility model compared with prior art has the following advantages:
The utility model is owing to be equipped with the reagent that is used to prepare miRNAs Poly (A) polysaccharase system in reagent bottle, be used to prepare the reagent of miRNAs reverse transcription reaction system, be used to prepare the reagent of miRNAs qPCR reaction system, the reagent that wherein is used to prepare miRNAs Poly (A) polysaccharase system comprises rATP reagent, poly (A) polysaccharase reagent, polyApolymerase Buffer reagent, DEPC water, the reagent that is used to prepare miRNAs reverse transcription reaction system comprises Universal RT Primer reagent, dNTP reagent, M-MLV reagent, RT Buffer reagent, RNasin Inhibitor reagent, DEPC water, the reagent that is used to prepare the miRNAsqPCR reaction system comprises Universal qPCR Primer reagent, SYBR GreenMaster Mix reagent, sterile purified water, the utility model just provides the reagent of the method for miRNAs content in the rapid detection different sources sample like this, with rATP is substrate, add poly (A) tail by polyA polymerase (PAP) at the 3 ' end of miRNAs, utilize the miRNAs reverse transcription of the Universal RT Primer of M-MLV RTase and unique design with poly Aization, utilizing special Universal RT Primer pairing primer and specific miRNAs primer at last, is that fluorescence dye carries out the detection of miRNAs fast quantification with SYBR Green I.This test kit adopts PAP method and SYBR fluorescent quantitative PCR technique to combine, and has therefore that suitability is wider, reaction is sensitiveer, characteristics of high duplication and high precision.
The utlity model has following characteristics: 1) the parent material requirement is few, and the total RNA sample that is low to moderate 10ng just can be used for this test kit and detect; 2) range of application is wider, reduces experimental cost greatly.The sample that uses the PAP method to handle can satisfy the requirement of the different miRNAs of batch detection, greatly reduces experimental cost; 3) high sensitivity, wider dynamic change scope.The strongest pcr amplification signal can be obtained by the suggested design use, the miRNAs (detecting the dynamic change of 7 number order magnitude range) that is low to moderate 10 copies can be detected; 4) detect more quickly, can be as short as in the 3h and just can obtain quality data.
Description of drawings
Fig. 1 is the utility model fast m iRNAs quantitative PCR detection kit structural representation.
Embodiment
Below in conjunction with accompanying drawing the utility model fast m iRNAs quantitative PCR detection kit is described in further detail.
As shown in Figure 1, the utility model fast m iRNAs quantitative PCR detection kit, comprise outter box 1, inner box 2, be used to prepare the reagent 3 of miRNAs Poly (A) polysaccharase system, be used to prepare the reagent 4 of miRNAs reverse transcription reaction system, be used to prepare the reagent 5 of miRNAsqPCR reaction system, inner box 2 is provided with bearing holes, the reagent 3 that wherein is used to prepare miRNAsPoly (A) polysaccharase system comprises rATP reagent, poly (A) polysaccharase reagent, polyA polymerase Buffer reagent, DEPC water, the reagent 4 that is used to prepare miRNAs reverse transcription reaction system comprises Universal RT Primer reagent, dNTP reagent, M-MLV reagent, RT Buffer reagent, RNasin Inhibitor reagent, DEPC water, the reagent 5 that is used to prepare miRNAs qPCR reaction system comprises Universal qPCR Primer reagent, SYBR Green Master Mix reagent, sterile purified water, DEPC water, aseptic distillation moisture and surplus each reagent thereof are loaded on respectively in the corresponding reagent bottle, and each reagent bottle is placed on respectively in the bearing holes on the inner box.Inner box 2 places in the outter box 1, and outter box 1 passes through thin-film package.Wherein Poly (A) polysaccharase promptly is polyA polymerase.
Working method
Required other materials also comprises
1.) sample: the microRNA 10ng of total RNA 10ng that the good purity of quality is high～10 μ g or purifying～2.5 μ g.
2.) reagent: extracting RNA need be used Trizol, the saturated phenol of Tris (pH 4.5), chloroform, Virahol, or RNA extraction agent box; The upstream primer (10 μ M) that is used for quantitative PCR.
3.) instrument: PCR instrument or constant temperature instrument, whizzer, quantitative PCR instrument.
4.) consumptive material: the rifle head of no RNase, DNase, centrifuge tube, pipettor etc.
1.miRNAs adding poly (A) tail handles
MiRNAs is the RNA of non-coding, does not have poly (A).This test kit adopts poly (A) polysaccharase (PAP) to add rATP one by one at miRNAs 3 ' end, forms poly (A) tail.
1. melt rATP and PAP reagent on ice, flick mixing, of short duration centrifugal be placed on stand-by on ice;
2. according to the form below is prepared poly (A) change system:
| Reagent | Volume/consumption | Final concentration |
| Total RNA | χμl(2μg) | 0.1μg/μl |
| rATP(10mM) | 1μl | 0.5mM |
| 10×Buffer | 2μl | 1× |
| PAP | 2μl | 1U |
| DEPC water | (16.8-χ)μl | |
| Amount to | 20μl |
3. add poly (A) end reaction:
Flick the mixing reaction mixture, of short durationly centrifugally be placed on 37 ℃ of reaction 30mi, and 95 ℃ of reaction 5min inactivators.It is synthetic that products therefrom can carry out cDNA first chain immediately, also in-20 ℃ of preservations.
Carry out the RNA relating operation, need strictness to prevent to pollute.The centrifuge tube of the experimental implementation that is useful on and rifle head all should be removed DNase, RNase and carry out high-temperature sterilization and handle; Experiment operator need wear masks and disposable rubber gloves; The preparation of all reaction solutions etc. all needs to finish on ice.Total initial consumption of RNA can miRNAs abundance per sample be adjusted between 10ng～10 μ g.Best total RNA consumption at 100ng between the 2.5 μ g.Total RNA purity OD
260/ OD
280(10mM Tris, pH 7.5) is worth measurement.Be used for total RNA OD that miRNAs detects
260/ OD
280Value is advisable between 1.6～2.0.
2. reverse transcription miRNAs
Under the effect of M-MLV reversed transcriptive enzyme, the miRNAs of Poly Aization is reversed record for having the strand cDNA of anchor series.The reverse transcription system of this test kit provides certified reagent, can carry out reverse transcription efficiently to total RNA or poly (A) RNA within 40min, and its product can be directly used in the qPCR reaction.
1. 10 μ l (1 μ g) Poly Aization RNA is added in the EP pipe of no RNase, and in 65 ℃ of incubation 5min.Reaction finishes and places on ice immediately.
Carry out the RNA relating operation, need strictness to prevent to pollute.The centrifuge tube of the experimental implementation that is useful on and rifle head all should be removed DNase, RNase and carry out high-temperature sterilization and handle; Experiment operator need wear masks and disposable rubber gloves; The preparation of all reaction solutions etc. all needs to finish on ice.As use the microRNA of purifying, its usage quantity can be adjusted between 10ng～2.5 μ g.
2. according to the form below is prepared the reverse transcription reaction system in proper order
| Reagent | Volume/consumption | Final concentration |
| Total RNA | 1μg/10μl | 0.5μg/μl |
| Universal RT Primer (10μM) | 1μl | 0.5μM |
| dNTP(10μM) | 2μl | 1μM |
| M-MLV | 0.5μl | 2.5 |
| 5×RT Buffer | 4μl | 1× |
| RNase Inhibitor | 0.5μl | 10-20U |
| DEPC water | 2μl | |
| Amount to | 20μl |
According to the consumption of RNA, reaction volume can wait multiplication to subtract.
3. RT reaction:
Flick the mixing reaction mixture, the of short duration centrifugal 42 ℃ of reaction 30min that are placed on.Reaction finishes the back in 95 ℃ of heating 5min termination reactions.If have plenty of time, adopt 30 ℃ of reaction 10min, follow the RT programs of 42 ℃ of reaction 30min, better effects if.
4. cDNA template dilution:
RT gained cDNA template is with 5 times of deionized water dilutions, flick mixing and of short duration centrifugal after, can carry out the miRNAs quantitative analysis immediately, but also packing is stored in-20 ℃.
3.miRNAs qPCR detect
This test kit adopts the chimeric fluorescent method of SYBR Green I to carry out the analysis of miRNAs real-time fluorescence quantitative PCR.SYBR Green I is a kind of fluorescence dye that is incorporated into the double-stranded DNA ditch.In the PCR reaction system, add excessive SYBR Green I fluorescence dye, after SYBR GreenI fluorescence dye mixes double-stranded DNA specifically, the emitting fluorescence signal, and the SYBR Green I dye molecule that does not mix in the chain can not launched any fluorescent signal, thereby the increase of the increase of assurance fluorescent signal and PCR product is synchronous fully.
1. miRNAs design of primers:
The sequence of the downstream primer that uses among the qPCR and reverse transcriptase primer complementary pairing are provided by this test kit.The specific amplification of miRNAs is mainly controlled by specific upstream primer, therefore will detect different miRNAs and should increase with different specificity upstream primers.This test kit does not provide specific upstream primer, need be designed voluntarily or be ordered by the client.
The upstream primer principle of design is: primer length should be not less than 15nt; According to the based composition of different miRNAs mature sequences, the complete mature sequence of general direct usefulness is as the upstream primer of qPCR, and some will and form the primer dimer tendency according to mature sequence GC content, suitably delete several bases at 3 ' end of mature sequence.
Upstream primer should guarantee partial sequence at least with miRNAs template 15nt pairing, still can efficiently synthesize the cDNA product in the case, and have the favorable linearity variation range.The primer that is lower than miRNAs length 12nt will cause high background Ct value.
3. prepare following qPCR reaction system on ice:
| Reagent | Volume (μ l) | Final concentration |
| The cDNA of dilution | 1 | |
| Upstream primer (10 μ M) | 0.5 | 0.2μM |
| Universal qPCR Primer(10μM) | 0.5 | 0.2μM |
| 2×SYBR Green Master Mix | 10 | |
| Sterile purified water | 8 | |
| Amount to | 20 |
The addition of cDNA template that is used for qPCR is usually below 100ng.The content difference that contains target miRNAs in different types of cDNA template can be carried out gradient dilution in case of necessity, to determine optimal Template concentration.When the concentration of primer was 0.2 μ M, the result who carries out qPCR was better.When reactivity worth was relatively poor, primer concentration can be adjusted between 0.1～1.0 μ M.Carry out the miRNAs quantitative PCR detection, suggestion is provided with other does not have the microRNA relevant, that expression is stable as confidential reference items, for example U6 snRNA, 5S rRNA, tRNA.Detect two confidential reference items and can make the more accurate and tool cogency of data.Sometimes miRNAs such as let-7a, miR-10b, miR-16, miR-21 also are used as the quantitative confidential reference items of miRNAs.When the miRNAs of breast cancer tissue was carried out detection by quantitative, adopting the two confidential reference items of let-7a and miR-16 was optimum combination.Proposal reactions liquid is long-pending to be 20～50 μ l.
4. carry out the qPCR reaction:
Set instrument parameter (is example with ABI-PRISM 7000/7700/7900HT instrument) in advance.Abundant mixing reaction mixture of short durationly places reaction tubes in the machine after centrifugal, thereby carries out the PCR reaction.Contain the ROX correction dye among the SYBR Green Master Mix, be used for the fluorescent signal error that produces between correction hole and the hole, can use on the Real-Time pcr amplification instrument of companies such as ABI, Stratagene, Roche.When using other instruments to carry out quantitative PCR analysis, please adjust reaction conditions according to the instrument explanation.Use SYBRGreen I dyestuff to carry out qPCR, need the solubility curve of monitoring product.Single PCR product has only a fusion peak.
Claims (4)
1, a kind of fast m iRNAs quantitative PCR detection kit, comprise outter box, inner box, inner box is provided with bearing holes, inner box places in the outter box, it is characterized in that: be placed with the reagent bottle group that the reagent that is used to prepare miRNAs Poly (A) polysaccharase system is housed in the bearing holes on the described inner box, the reagent bottle group of the reagent that is used to prepare miRNAs reverse transcription reaction system is housed and the reagent bottle group of the reagent that is used to prepare miRNAs qPCR reaction system is housed, the reagent bottle group that the reagent that is used to prepare miRNAs Poly (A) polysaccharase system wherein is housed comprises the reagent bottle that rATP reagent is housed, the reagent bottle of poly (A) polysaccharase reagent is housed, the reagent bottle and the reagent bottle that DEPC water is housed of polyA polymeraseBuffer reagent are housed, the reagent bottle group that the reagent that is used to prepare miRNAs reverse transcription reaction system is housed comprises the reagent bottle that Universal RT Primer reagent is housed, the reagent bottle of dNTP reagent is housed, the reagent bottle of M-MLV reagent is housed, the reagent bottle of RTBuffer reagent is housed, the reagent bottle of RNasin Inhibitor reagent is housed and the reagent bottle of DEPC water is housed, the reagent bottle group that the reagent that is used to prepare miRNAs qPCR reaction system is housed comprises the reagent bottle that Universal qPCR Primer reagent is housed, the reagent bottle and the reagent bottle that sterile purified water is housed of SYBR Green MasterMix reagent are housed.
2. fast m iRNAs quantitative PCR detection kit according to claim 1, it is characterized in that: the volume specification of the rATP in the reagent bottle of the described rATP of being equipped with reagent is 10 μ l, the polyA polymerase volume specification that is equipped with in the reagent bottle of poly (A) polysaccharase reagent is 20 μ l, the RNasin Inhibitor volume specification that is equipped with in the reagent bottle of RNasin Inhibitor reagent is 10 μ l, the DEPC volume of water specification that is equipped with in the reagent bottle of DEPC water is 250 μ l, the M-MLV volume specification that is equipped with in the reagent bottle of M-MLV reagent is 10 μ l, the Universal qPCR Primer volume specification that is equipped with in the reagent bottle of UniversalqPCR Primer reagent is 50 μ l, the Universal RT Primer volume specification that is equipped with in the reagent bottle of Universal RT Primer reagent is 20 μ l, the SYBRGreen Master Mix volume specification that is equipped with in the reagent bottle of SYBR Green Master Mix reagent is 1000 μ l, the dNTP volume specification that is equipped with in the reagent bottle of dNTP reagent is 40 μ l, the aseptic distillation volume of water specification that is equipped with in the reagent bottle of sterile purified water is 1000 μ l, the RT Buffer 5 * RT Buffer volume specification that wherein is equipped with in the reagent bottle of RT Buffer reagent is 80 μ l, the polyA polymerase Buffer 10 * polyA polymerase Buffer volume specification that is equipped with in the reagent bottle of polyA polymerase Buffer reagent is 20 μ l, and the reagent bottle that contains each reagent is the unified specification size.
3. fast m iRNAs quantitative PCR detection kit according to claim 1, it is characterized in that: the described reagent bottle group that the reagent that is used to prepare miRNAs Poly (A) polysaccharase system is housed all also comprises the reagent bottle that noncoding total RNA is housed, the OD of total RNA with the reagent bottle group that the reagent that is used to prepare miRNAs reverse transcription reaction system is housed
260/ OD
280Value is between 1.6～2.0, and the reagent bottle group that the reagent that is used to prepare miRNAs qPCR reaction system is housed also comprises the reagent bottle of the cDNA that dilution is housed and the reagent bottle of miRNA upstream primer is housed.
4. according to claim 2 or 3 described fast m iRNAs quantitative PCR detection kits, it is characterized in that: described outter box passes through thin-film package.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676523A (en) * | 2012-05-16 | 2012-09-19 | 北京旷博生物技术有限公司 | Breast cancer molecular marker miR-139-5p |
| CN102817085A (en) * | 2012-04-26 | 2012-12-12 | 深圳北京大学香港科技大学医学中心 | Method for rapid reverse transcription of micro ribonucleic acid (RNA) library |
-
2009
- 2009-03-13 CN CN200920052584U patent/CN201381322Y/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102817085A (en) * | 2012-04-26 | 2012-12-12 | 深圳北京大学香港科技大学医学中心 | Method for rapid reverse transcription of micro ribonucleic acid (RNA) library |
| CN102817085B (en) * | 2012-04-26 | 2014-07-16 | 深圳北京大学香港科技大学医学中心 | Method for rapid reverse transcription of micro ribonucleic acid (RNA) library |
| CN102676523A (en) * | 2012-05-16 | 2012-09-19 | 北京旷博生物技术有限公司 | Breast cancer molecular marker miR-139-5p |
| CN102676523B (en) * | 2012-05-16 | 2013-05-29 | 北京旷博生物技术有限公司 | Breast cancer molecular marker miR-139-5p |
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Granted publication date: 20100113 Termination date: 20120313 |