CN201381321Y - Fast miRNAs stem loop method detection kit - Google Patents
Fast miRNAs stem loop method detection kit Download PDFInfo
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- CN201381321Y CN201381321Y CN200920052582U CN200920052582U CN201381321Y CN 201381321 Y CN201381321 Y CN 201381321Y CN 200920052582 U CN200920052582 U CN 200920052582U CN 200920052582 U CN200920052582 U CN 200920052582U CN 201381321 Y CN201381321 Y CN 201381321Y
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Abstract
The utility model discloses a fast miRNAs stem-loop method detection kit, comprising an outer box body and an inner box body. Bearing holes are arranged on the inner box body. The fast miRNAs stem loop method detection kit is characterized in that an miRNAs quantification PCR detection kit also comprises a reagent used for preparing a reverse transcription reaction system and a reagent used for preparing a PCR reaction system, wherein the reagent used for preparing the reverse transcription reaction system comprises a Stem-loop RTPrimer reagent, a dNTP reagent, an M-MLV reagent, an RT Buffer reagent, an Rnase Inhibitor reagent and DEPC water, the reagent used for preparing the PCR reaction system comprises a Specific Forward Primer reagent, a Stem-loop qPCR Primer reagent, an SYBR Green Master Mix reagent and sterilization distilled water, the DEPC water, the sterilization distilled water and the residual reagents are respectively installed 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 stronger specificity, more accurate result and better repeatability.
Description
Technical field
The utility model relates to the around-France detection kit of a kind of fast m iRNAs stem.
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 specificity is stronger, the result is more accurate, a better around-France detection kit of fast m iRNAs stem of repeatability.
The purpose of this utility model is achieved through the following technical solutions.
The around-France detection kit of a kind of fast m iRNAs stem, 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 the reverse transcription reaction system is housed in the bearing holes on the described inner box, the reagent bottle group of the reagent that is used to dispose the PCR reaction system is housed, the reagent bottle group that the reagent that is used to prepare the reverse transcription reaction system wherein is housed comprises the reagent bottle that Stem-loop 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 RNase Inhibitor reagent is housed, the reagent bottle of DEPC water is housed, and the reagent bottle group that the reagent that is used to dispose the PCR reaction system is housed comprises the reagent bottle that Specific Forward Primer reagent is housed, the reagent bottle of Stem-loopqPCR Primer reagent is housed, the reagent bottle of SYBR Green Master Mix reagent is housed, the reagent bottle of sterile purified water is housed.
The volume specification of M-MLV in the reagent bottle of the described M-MLV of being equipped with reagent is 5 μ l, the RNase Inhibitor volume specification that is equipped with in the reagent bottle of RNase Inhibitor reagent is 5 μ l, the Stem-loopqPCR Primer volume specification that is equipped with in the reagent bottle of Stem-loop qPCR Primer reagent is 20 μ l, the dNTP volume specification that is equipped with in the reagent bottle of dNTP reagent is 40 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 Stem-loop qPCR Primer volume specification that is equipped with in the reagent bottle of Stem-loop qPCRPrimer reagent is 50 μ l, the Specific ForwardPrimer volume specification that is equipped with in the reagent bottle of Specific Forward Primer reagent is 50 μ l, the DEPC volume of water specification that is equipped with in the reagent bottle of DEPC water is 500 μ l, the aseptic distillation volume of water specification that is equipped with in the reagent bottle of sterile purified water is 1ml, and the RT Buffer 5 * RT Buffer volume specification that wherein is equipped with in the reagent bottle of RT Buffer reagent is 40 μ l.
The described reagent bottle group that the reagent that is used to prepare the reverse transcription reaction system is housed also comprises the reagent bottle that noncoding total RNA is housed, the OD of total RNA
260/ OD
280Value is between 1.6～2.0, and the reagent bottle group that the reagent that is used to dispose the PCR reaction system is housed also comprises the reagent bottle that cDNA reagent 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 the reverse transcription reaction system in reagent bottle, be used to dispose the reagent of PCR reaction system, the reagent that wherein is used to prepare the reverse transcription reaction system comprises Stem-loop RT Primer reagent, dNTP reagent, M-MLV reagent, RTBuffer reagent, RNase Inhibitor reagent, DEPC water, the reagent that is used to dispose the PCR reaction system comprises Specific Forward Primer reagent, Stem-loop qPCRPrimer 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, each reagent bottle is placed on respectively in the bearing holes on the inner box, and the utility model just provides the method for miRNAs content in the rapid detection different sources sample like this.This test kit utilizes M-MLV RTase and unique design reverse transcriptase primer (Stem-loop RT Primer), with miRNAs reverse transcription specific in the sample is the cDNA that 3 ' end has the loop-stem structure joint, and utilizing miRNAs Auele Specific Primer (Stem-loop qPCR Forward Primer) and stem ring joint pairing primer (Stem-loop qPCR Reverse Primer) at last is that fluorescence dye carries out the detection of miRNAs fast quantification with SYBR Green I.This test kit adopts around-France reverse transcription of stem and SYBR quantitative PCR technique to combine, and has therefore that specificity is stronger, the result is more accurate, better characteristics of repeatability.
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) high specificity, more pinpoint accuracy; 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 around-France detection kit structural representation of the utility model fast m iRNAs stem.
Embodiment
Below in conjunction with accompanying drawing the around-France detection kit of the utility model fast m iRNAs stem is described in further detail.
As shown in Figure 1, the around-France detection kit of the utility model fast m iRNAs stem, comprise outter box 1, inner box 2, be used to prepare the reagent 3 of reverse transcription reaction system, be used to dispose the reagent 4 of PCR reaction system, inner box 2 is provided with bearing holes 21, the reagent 3 that wherein is used to prepare the reverse transcription reaction system comprises Stem-loop RT Primer reagent, dNTP reagent, M-MLV reagent, RT Buffer reagent, RNase Inhibitor reagent, DEPC water, the reagent 4 that is used to dispose the PCR reaction system comprises Specific Forward Primer reagent, Stem-loop 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 single reagent bottle, and each reagent bottle is placed on respectively in the bearing holes 21 on the inner box 2.Inner box 2 places in the outter box 1, and outter box 1 passes through thin-film package.
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. 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. the total RNA of 2 μ g 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.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 1.0 μ g.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 | χμl(2μg) | 0.1μg/μl |
| Stem-loop RT Primer (10μM) | 1μl | 0.5μM |
| dNTP(10μM) | 2μl | 1μM |
| M-MLV | 0.5μl | 2.5U |
| 5×RT Buffer | 4μl | 1× |
| RNase Inhibitor | 0.5μl | 10-20U |
| DEPC water | (12-χ)μl | |
| Amount to | 20μl |
According to the consumption of RNA what, 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 ℃.
2.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. dispose following PCR reaction system on ice:
| Reagent | Volume (μ l) | Final concentration |
| cDNA | 1 | |
| Specific Forward Primer(10μM) | 0.5 | 0.25μM |
| Stem-loop qPCR Primer(10μM) | 0.5 | 0.25μM |
| 2×SYBR Green Master Mix | 10 | 1× |
| 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.Proposal reactions liquid is long-pending to be 20～50 μ l.
2. 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, carries out the PCR reaction then.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, the around-France detection kit of a kind of fast m iRNAs stem, 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 the reverse transcription reaction system is housed in the bearing holes on the described inner box, the reagent bottle group of the reagent that is used to dispose the PCR reaction system is housed, the reagent bottle group that the reagent that is used to prepare the reverse transcription reaction system wherein is housed comprises the reagent bottle that Stem-loop 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 RNase Inhibitor reagent is housed, the reagent bottle of DEPC water is housed, and the reagent bottle group that the reagent that is used to dispose the PCR reaction system is housed comprises the reagent bottle that SpecificForward Primer reagent is housed, the reagent bottle of Stem-loop qPCR Primer reagent is housed, the reagent bottle of SYBR Green Master Mix reagent is housed, the reagent bottle of sterile purified water is housed.
2. the around-France detection kit of fast m iRNAs stem according to claim 1, it is characterized in that: the volume specification of the M-MLV in the reagent bottle of the described M-MLV of being equipped with reagent is 5 μ l, the RNase Inhibitor volume specification that is equipped with in the reagent bottle of RNase Inhibitor reagent is 5 μ l, the Stem-loop qPCRPrimer volume specification that is equipped with in the reagent bottle of Stem-loop qPCR Primer reagent is 20 μ l, the dNTP volume specification that is equipped with in the reagent bottle of dNTP reagent is 40 μ l, the SYBR GreenMaster Mix volume specification that is equipped with in the reagent bottle of SYBR Green Master Mix reagent is 1000 μ l, the Stem-loop qPCR Primer volume specification that is equipped with in the reagent bottle of Stem-loop qPCR Primer reagent is 50 μ l, the Specific Forward Primer volume specification that is equipped with in the reagent bottle of SpecificForward Primer reagent is 50 μ l, the DEPC volume of water specification that is equipped with in the reagent bottle of DEPC water is 500 μ l, the aseptic distillation volume of water specification that is equipped with in the reagent bottle of sterile purified water is 1ml, and the RT Buffer 5 * RT Buffer volume specification that wherein is equipped with in the reagent bottle of RTBuffer reagent is 40 μ l.
3. the around-France detection kit of fast m iRNAs stem according to claim 1 is characterized in that: the described reagent bottle group that the reagent that is used to prepare the reverse transcription reaction system is housed also comprises the reagent bottle that noncoding total RNA is housed, the OD of total RNA
260/ D
280Value is between 1.6～2.0, and the reagent bottle group that the reagent that is used to dispose the PCR reaction system is housed also comprises the reagent bottle that cDNA reagent is housed.
4. according to claim 2 or the around-France detection kit of 3 described fast m iRNAs stems, it is characterized in that: described outter box passes through thin-film package.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200920052582U CN201381321Y (en) | 2009-03-13 | 2009-03-13 | Fast miRNAs stem loop method detection kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200920052582U CN201381321Y (en) | 2009-03-13 | 2009-03-13 | Fast miRNAs stem loop method detection kit |
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| CN201381321Y true CN201381321Y (en) | 2010-01-13 |
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| CN200920052582U Expired - Fee Related CN201381321Y (en) | 2009-03-13 | 2009-03-13 | Fast miRNAs stem loop method detection kit |
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Legal Events
| Date | Code | Title | Description |
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| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20100113 Termination date: 20120313 |