CN103205489A - Detection method for expression of MicroRNA and quantitative detection kit for MicroRNA - Google Patents
Detection method for expression of MicroRNA and quantitative detection kit for MicroRNA Download PDFInfo
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Abstract
The invention discloses a detection method for expression of MicroRNA and a quantitative detection kit for MicroRNA. The method comprises the following steps: (1) extracting MicroRNA of a sample and connecting the MicroRNA to a Poly A tail; (2) mixing three primers with a stem-loop structure and then carrying out annealing treatment; (3) subjecting the MicroRNA connected with the Poly A tail and the primers with the stem-loop structure to annealing connection; (4) carrying out inverse transcription with the MicroRNA as a template so as to obtain cDNA; and (5) with the cDNA as a template, adding primers of an amplification target MicroRNA and detecting expression of the target MicroRNA by using a fluorescence quantitative PCR method. According to the detection method, all the MicroRNA and reference genes of a to-be-detected object can be obtained through one inverse transcription, so generation of a primer dimer and a complex hairpin structure is substantially reduced, and detection efficiency of MicroRNA is greatly improved.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of Micro rna expression detection method and detection by quantitative test kit thereof.
Background technology
Micro RNA is the non-coding single stranded RNA on the class source intrinsic staining body of finding in multiple eukaryotic cell and virus in recent years, length is the short sequence of 21～25nt, the conservative property that has height in evolution, can be by matching with the specific base complementrity of said target mrna, cause the said target mrna degraded or suppress its translation, thereby expression regulation (the O`Donnell KA after gene transcribed, Wentzel EA, Zeller KI, et al.e-Myc-regulated microRNAs modulate E2F1 expression[J] .Nature, 2005,4 (7043): 839.).Because Micro RNA sequence has certain conservative property in different biologies, the function that generally believes Micro RNA at present is some primary processes that participate in life, as the cell proliferation in the growth course, necrocytosis, stress reaction and metabolism of fat etc., more and more evidences shows that Micro RNA plays the vital role of regulate gene expression in cell.The nearest canceration of discovering Micro RNA and cell has extremely close relation, have been found that Micro RNA relevant with tumor diseases such as lung cancer, colorectal carcinoma, Burkitt lymphoma and chronic lymphocytic leukemias (Meltzer PS.Cancer genemics:Small RNAs with big impacts[J] .Nature, 2005,435 (7043): 745.).
Because Micro RNA molecule has only size about 20-25nt, detect comparatively difficulty.The main method that adopts was that the method for hybridization such as Northern blot detects in the past, and method is loaded down with trivial details, be difficult for successfully, and Micro RNA very easily degraded, and influenced also bigger for the result.
Defective for detection method before overcoming, Allawi etc. have set up method (the Allawi HT of Invader Assay, Deahlberg JE, OlsonS, et al.Quantitation of Micro RNAs using a modified Invader assay.RNA, 2004,10:1153-1161), Liu etc. have set up oligonucleotide chip method (Liu C, Calmn GA, et al.An oligo nucleotide microchipfor genome-wide micro RNA profiling in human and mouse tissues.PNAS, 2004,101 (26): 9740-9744), this method has improved the susceptibility and the practicality that detect, but this method needs fluorescent mark and luminoscope or chip detector, and because micro RNA molecule is too little, uses and also be subjected to certain restriction.
Nowadays, detection to Micro RNA precursor mainly is method (the Schmittgen TD that adopts RT-PCR, Jiang J, Liu Q, et al.A high-throughput method to monitor the expression of Micro RNA precursors.Nucleic Acids Research.2004,32 (4): e43), adopt random primer or directly carry out reverse transcription with the primer of Micro RNA precursor complementation, but this can only be at special Micro RNA, and semiquantitative method can not show the expression amount of Micro RNA exactly, so this method has withdrawed from the ranks of the detection of Micro RNA molecule gradually.
Aforesaid method when reverse transcription Micro RNA, all seem too loaded down with trivial details and efficient not high, so a kind of " tailing method " occurred, namely prolong and adopt the method for real-time PCR that Micro RNA is detected at the 3 ' end of Micro RNA.Common tailing method has two kinds: a kind of is to add Poly-A tail (Shi R by terminal enzyme (DNA) at the 3` end of Micro RNA molecule, Chiang V L.Facile means for quantifying Micro RNA expression by real-time PCR[J] .BioTechniques, 2005,39 (4): 519-525.), Oligo-dT with 30～40 Nucleotide carries out the cDNA that reverse transcription obtains being extended as primer, can use SYBR Green to carry out real-time quantitative PCR and detect; Another kind is directly to use with the long primer reverse transcription that adds of the terminal pairing of Micro RNA to become cDNA, then use primer that LNA modifies by Real-time PCR method the expression of Micro RNA to be detected (Raymond C K, Roberts B S, Garrett-engele P.et al, Simple quantitative primer-extension PCR for direct monitoring of Micro RNA and short-interfering RNAs[J] .RNA, 2005.11 (11): 1737-1744.).Yet in these two kinds of methods, the cDNA that is synthesized of tailing method is too short relatively for PCR at random, only be fit to use the taq man method of probe to detect, and add the method for the terminal pairing of specificity micro RNA afterbody, can only synthesize the specific cDNA of Micro RNA, can't reverse simultaneously with confidential reference items, may cause bigger error.
In order to detect the expression amount of Micro RNA more efficiently, and raising accuracy, (Chen C such as Chen, Ridzon D A, Broomer A J, et al.Realtime quantification of micro RNAs by stem-loop RT-PCR[J] .Nucleic Acids Res, 2005,33 (20): 179.) on the basis that adds the long primer reverse transcription of the terminal pairing of specificity micro RNA, real time round pcr is incorporated in the middle of the detection of micro RNA, has set up the reverse transcription quantitative PCR technique (stem-loop RT-PCR) of stem ring by name.He has at first designed a probe with reverse transcription of loop-stem structure, adopts the probe technique of Taq man then, and Micro RNA is detected, and it is reported that this structure has improved efficient and the specificity of Micro RNA reverse transcription greatly.But also caused high specific the time once counter-rotating reverse transcription to go out a special Micro RNA like this, are a kind of great restrictions for detecting whole Micro RNA and the expression situation of other small fragment RNAs, use the method for Taqman probe also to improve the detection cost of Micro RNA greatly simultaneously.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is exactly to detect comparatively difficulty at Micro RNA developed by molecule, the conventional detection complex steps, be difficult for successfully, and gained Micro RNA very easily degrades, the result there is the defective of considerable influence, a kind of Micro rna expression detection method and a kind of Micro RNA detection by quantitative test kit are provided.
For solving the problems of the technologies described above, one of technical scheme that the present invention takes is: a kind of Micro rna expression detection method, and wherein said method comprises:
(1) the Micro RNA of extraction sample connects the PolyA tail with gained Micro RNA;
(2) carry out anneal after three primers with loop-stem structure are mixed, the sequence of described primer with loop-stem structure is respectively shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ IDNO:3 in the sequence table;
(3) the Micro RNA that step (1) gained is connected with Poly A tail is connected with the primer annealing that step (2) gained has loop-stem structure;
(4) be that the template reverse transcription obtains cDNA with step (3) gained Micro RNA;
(5) be template with step (4) gained cDNA, add the primer of amplification target Micro RNA, detect the expression of target Micro RNA by fluorescence quantifying PCR method.
Wherein the method for the described extraction sample of step (1) Micro RNA is the ordinary method of this area.The extraction of described sample Micro RNA can utilize various commercial reagent box to carry out.The method that described Micro RNA connects the PolyA tail is the conventional tailing method of using in this area, the method of the Poly of adding A tail of the present invention preferably comprises: add Poly A polysaccharase, add ATP solution, with 35～37 ℃ of reactions of this mixed solution, 10～15min namely.
Wherein described three primers with loop-stem structure of step (2) are the conventional primer in this area.The sequence of described three primers with loop-stem structure is preferably respectively shown in SEQ ID NO:1, SEQ ID NO:2 in the sequence table and SEQ ID NO:3.Wherein said preparation method with primer of loop-stem structure is the preparation method of this area routine, and of the present invention have the primer of loop-stem structure preferably for synthetic namely by artificial complete sequence.
Wherein the described anneal of step (2) is the anneal technology of this area routine.The program of anneal of the present invention preferably is: (1) 95 ℃ of reaction 2min, and (2) descended 0.1 ℃ in per 5 ~ 10 seconds, were down to 35 ℃ ~ 15 ℃, (3) 4 ℃ of preservations more preferably are (1) 95 ℃ of reaction 2min, and (2) descended 0.1 ℃ in per 8 seconds, be down to 25 ℃, (3) 4 ℃ of preservations.
Wherein the program of the described annealing connection of step (3) preferably is: 60～70 ℃, and reaction 5～10min, namely.
Wherein the described reverse transcription of step (4) is the conventional reverse transcription technology in this area, and the program of reverse transcription of the present invention preferably is: (1) 30 ℃ was reacted 10 ~ 15 minutes; (2) 42 ℃ were reacted 50 ~ 60 minutes; (3) 50 ℃ were reacted 10 ~ 15 minutes; (4) 95 ℃ were reacted 10 ~ 15 minutes; (5) with gained cDNA sample in 4 ℃ of preservations.
Wherein the primer of the described amplification target of step (5) Micro RNA is: the general reverse primer of Micro RNA and Micro RNA detect forward primer.
The general reverse primer of wherein said Micro RNA is the general reverse primer of Micro RNA of this area routine, the general reverse primer sequence of Micro RNA of the present invention preferably is: 5 '-CTGCAGTGGCGGACCA-3 ', and shown in SEQ ID NO:4 in the sequence table.
Wherein said Micro RNA detects the Micro RNA detection forward primer that forward primer is this area routine, Micro RNA of the present invention detects forward primer: Has-MiR-365a detects forward primer: 5 '-TAATGCCCCTAAAAATCCTTAT-3 ', and this primer sequence is shown in SEQ ID NO:5 in the sequence table.The preparation method of the primer of described amplification target Micro RNA is the conventional preparation method in this area, and is preferably synthetic namely for artificial complete sequence.
Wherein the described quantitative fluorescent PCR of step (5) is the conventional fluorescent quantitative PCR technique that uses in this area (or be called real time round pcr, abbreviate qPCR or qRT-PCR as).Can adopt different quantitative fluorescent PCR programs to detect at different target Micro RNA.The program of quantitative fluorescent PCR of the present invention preferably is: 95 ℃ of (1) pre-sex change, 1 minute; (2) sex change is 95 ℃, 5S; (3) annealing is extended 60 ℃, 20S; Step (2)～(3) are carried out 40 circulations altogether, read light absorption value in the extension stage at every turn.
For solving the problems of the technologies described above, two of the technical scheme that the present invention takes is: a kind of primer with loop-stem structure, the sequence of described primer with loop-stem structure is shown in SEQ ID NO:1, SEQ ID NO:2 in the sequence table or SEQ ID NO:3.
Wherein said primer with loop-stem structure is the primer with loop-stem structure of this area routine, and the primer with loop-stem structure of the present invention preferably is:
Oligo1：
5 '-GTCGGAGAGACTGCAGTGGCGGACCAATTCGCACTCTCTCCGACTTTTTTTTA-3 ' (shown in SEQ ID NO:1 in the sequence table);
Oligo2：
5 '-GTCGGAGAGACTGCAGTGGCGGACCAATTCGCACTCTCTCCGACTTTTTTTTC-3 ' (shown in SEQ ID NO:2 in the sequence table);
Oligo3：
5 '-GTCGGAGAGACTGCAGTGGCGGACCAATTCGCACTCTCTCCGACTTTTTTTTG-3 ' (shown in SEQ ID NO:3 in the sequence table).
Wherein said preparation method with primer of loop-stem structure is conventional preparation method, the primer with loop-stem structure of the present invention preferably for the complete sequence synthetic namely.
For solving the problems of the technologies described above, three of the technical scheme that the present invention takes is: a kind of Micro RNA detection by quantitative test kit, this Micro RNA detection by quantitative test kit comprises: SYBR Green I fluorescence dye, PCR damping fluid, dNTPs solution, archaeal dna polymerase, aseptic double-distilled water, the primer with neck ring structure, general reverse primer and Micro RNA detect forward primer, and the sequence of wherein said primer with neck ring structure is respectively shown in SEQ ID NO:1, SEQ ID NO:2 and SEQID NO:3 in the sequence table.
Wherein said SYBR Green I fluorescence dye, PCR damping fluid, dNTPs solution, archaeal dna polymerase, aseptic double-distilled water are the commercially available experiment reagent of this area routine.
Wherein said general reverse primer is the general reverse primer of this area routine.The general reverse primer sequence of Micro RNA of the present invention preferably is: 5 '-CTGCAGTGGCGGACCA-3 ', and shown in SEQ ID NO:4 in the sequence table.
Wherein said Micro RNA detects the Micro RNA detection forward primer that forward primer is this area routine, Micro RNA of the present invention detects forward primer preferably for Has-MiR-365a detects forward primer: 5 '-TAATGCCCCTAAAAATCCTTAT-3 ', and its sequence is shown in SEQ IDNO:5 in the sequence table.
The sequence of wherein said primer with neck ring structure is preferably respectively shown in SEQ IDNO:1, SEQ ID NO:2 in the sequence table and SEQ ID NO:3.The preparation method of wherein said primer is the conventional preparation method in this area, preferably prepares namely for synthetic.
Wherein said Micro RNA detection by quantitative test kit comprises also that preferably RNA extracts reagent and/or reverse transcription reagent.Wherein said RNA extracts reagent: Trizol reagent, RNA enzyme inhibitors.Wherein said reverse transcription reagent preferably is: reversed transcriptive enzyme (AMV), AMV damping fluid.Wherein said AMV damping fluid preferably comprises: dNTPs, random primer, RNA enzyme inhibitors.The preparation method of described reagent is the conventional preparation method in this area, preferably is commercially available gained.
Wherein said Micro RNA detection by quantitative test kit preferably also comprises the confidential reference items primer.The confidential reference items primer that confidential reference items primer of the present invention is this area routine, confidential reference items primer of the present invention preferably is U6-F and U6-R primer, the sequence of described confidential reference items primer is as follows:
U6-F:5 '-CTCGCTTCGGCAGCACA-3 ' is shown in SEQ ID NO:6 in the sequence table;
U6-R:5 '-AACGCTTCACGAATTTGCGT-3 ' is shown in SEQ ID NO:7 in the sequence table.Described confidential reference items method for preparing primer is the conventional preparation method in this area, preferably is artificial synthesized sequence.
The raw material that the present invention is used or reagent except specifying, equal commercially available getting.
Than prior art, beneficial effect of the present invention is as follows:
1, Micro rna expression detection method provided by the invention is utilized described primer with loop-stem structure, almost can obtain Micro RNA samples all in the testing sample by a reverse transcription, and internal control gene (U6 gene, this gene is the mRNA about 100bp), improved the detection efficiency of Micro RNA greatly.
2, the primer with loop-stem structure provided by the invention after the annealed processing, can significantly reduce the generation of primer dimer and complicated hairpin structure, thereby maximum amplitude ground has improved the counter-rotating efficient of this primer for Micro RNA.
Micro RNA detection by quantitative test kit of the present invention utilizes described primer with neck ring structure, can pass through a reverse transcription reaction, finish the reverse transcription that comprises confidential reference items and Micro RNA to be checked, greatly reduce the substep systematic error that the small segment mRNA of Micro RNA and confidential reference items produces of reversing, this method is simply efficient, thereby has significantly saved time and cost.
Description of drawings
Fig. 1 (A) is Cal-27 cell qPCR internal control gene (U6) amplification curve; Fig. 1 (B) is Cal-27 cell qPCR internal control gene (U6) melting curve figure; Fig. 1 (C) is Cal-27 cell qPCR gene to be checked (MiR-365a) amplification curve; Fig. 1 (D) is Cal-27 cell qPCR gene to be checked (MiR-365a) melting curve figure.Wherein a repeats 3 for the Cal-27 cell sample repeats 2, c for the Cal-27 cell sample for the Cal-27 cell sample repeats 1, b.
Fig. 2 (A) is Hep-2 cell qPCR internal control gene (U6) amplification curve; Fig. 2 (B) is Hep-2 cell qPCR internal control gene (U6) melting curve figure; Fig. 2 (C) is Hep-2 cell qPCR gene to be checked (MiR-365a) amplification curve; Fig. 2 (D) is Hep-2 cell qPCR gene to be checked (MiR-365a) melting curve figure.Wherein d repeats 3 for the Hep-2 cell sample repeats 2, f for the Hep-2 cell sample for the Hep-2 cell sample repeats 1, e.
Fig. 3 (A) is LOVO cell qPCR internal control gene (U6) amplification curve; Fig. 3 (B) is LOVO cell qPCR internal control gene (U6) melting curve figure; Fig. 3 (C) is LOVO cell qPCR gene to be checked (MiR-365a) amplification curve; Fig. 3 (D) is LOVO cell qPCR gene to be checked (MiR-365a) melting curve figure.Wherein g repeats 3 for the LOVO cell sample repeats 2, i for the LOVO cell sample for the LOVO cell sample repeats 1, h.
Fig. 4 (A) is MCF-7 cell qPCR internal control gene (U6) amplification curve; Fig. 4 (B) is MCF-7 cell qPCR internal control gene (U6) melting curve figure; Fig. 4 (C) is MCF-7 cell qPCR gene to be checked (MiR-365a) amplification curve; Fig. 4 (D) is MCF-7 cell qPCR gene to be checked (MiR-365a) melting curve figure.Wherein j repeats 3 for the MCF-7 cell sample repeats 2, l for the MCF-7 cell sample for the MCF-7 cell sample repeats 1, k.
Fig. 5 is Micro RNA365a basal expression detected result figure in Cal-27, Hep-2, LOVO, the MCF-7 clone.
Fig. 6 is the Micro RNA365a expression amount detected result figure of four kinds of clones (Cal-27, Hep-2, LOVO, MCF-7) among the comparative example 1.
Embodiment
Key instrument is: the desk-top high-speed refrigerated centrifuge of 5417R, thermo company, article number cat#5417R;
The PCR instrument, cat#2500;
ThermoForma310 series direet-heating type CO
2Incubator, thermo company, cat#3111;
Microplate reader Biotek company, VE-186;
Main agents is: PrimerScript ThermoScript II, Takara company, article number: cat#U1240;
E.coli Poly (A) polysaccharase, NEB company, article number: #M0276S;
5 * annealing buffer, green skies company, article number: D0251;
GXD kit iqSYBR Green, Bio-Rad company, article number: 1708882AP;
The Cal-27 cell strain, available from ATCC, article number: CRL-2095;
The Hep2 cell strain, available from Chinese Academy of Sciences's cell bank, article number: TCHu21;
The LOVO cell strain, available from Chinese Academy of Sciences's cell bank, article number: TCHu82;
The MCF-7 cell strain, available from Chinese Academy of Sciences's cell bank, catalog number (Cat.No.): TCHu74;
Improvement RPMI-1640 substratum, available from Hyclone, article No.: SH30809.01B;
FBS, foetal calf serum is available from the general biology that flies, article No.: 1101-500;
Penicillin-Streptomycin solution SP is two anti-, available from Hyclone, and article No.: J121071;
Micro RNA extraction agent box: Micro RNApure Mini kit is ShiJi Co., Ltd available from health, article No.: CW0627.
Embodiment 1Micro RNA reverse transcription detects primer
The present invention is directed to Micro RNA 3 primers with loop-stem structure are provided, described stem ring primer title and sequence are as follows respectively:
Oligo1：
5 '-GTCGGAGAGACTGCAGTGGCGGACCAATTCGCACTCTCTCCGACTTTTTTTTA-3 ' (shown in SEQ ID NO:1 in the sequence table);
Oligo2：
5 '-GTCGGAGAGACTGCAGTGGCGGACCAATTCGCACTCTCTCCGACTTTTTTTTC-3 ' (shown in SEQ ID NO:2 in the sequence table);
Oligo3：
5 '-GTCGGAGAGACTGCAGTGGCGGACCAATTCGCACTCTCTCCGACTTTTTTTTG-3 ' (shown in SEQ ID NO:3 in the sequence table).
Above-mentioned primer is prepared by the JaRa biosynthesizing.
The present invention has also utilized the general reverse primer of Micro RNA and Has-MiR-365a to detect forward primer, and described primer is as follows respectively:
The general reverse primer sequence of Micro RNA: 5 '-CTGCAGTGGCGGACCA-3 ' (shown in SEQ ID NO:4 in the sequence table);
Has-MiR-365a detects forward primer: 5 '-TAATGCCCCTAAAAATCCTTAT-3 ' (shown in SEQ ID NO:5 in the sequence table).
The confidential reference items primer that the present invention uses is U6-F and U6-R primer, and described primer is as follows:
U6-F:5 '-CTCGCTTCGGCAGCACA-3 ' (shown in SEQ ID NO:6 in the sequence table);
U6-R:5 '-AACGCTTCACGAATTTGCGT-3 ' (shown in SEQ ID NO:7 in the sequence table).
Above-mentioned primer is by JaRa biosynthesizing preparation, and the concentration of described primer is 100nmol/ μ l.
The preparation that embodiment 2 tests with each clone
Aseptic 37 ℃, 5%CO
2Under the condition, use the 1640+10%FBS+1%SP substratum to carry out cell attachment and cultivate, cultivate Cal-27, Hep2, LOVO, MCF-7 cell respectively, in the 6cm culture dish, treat that cell grows to about 80% after, can carry out extracting Micro RNA experiment.
The preparation of embodiment 3Micro RNA sample
1. single-layer culturing cell: inhale and remove nutrient solution, add Buffer RLM(Micro RNA extraction buffer, Lot:04911C; Be century available from health, be contained in Micro RNA and extract in the test kit, Cat:CW0627), every 10cm
2Add 1ml Buffer RLM.
2. blow and beat several times repeatedly after adding Buffer RLM in the sample, make its abundant cracking.Room temperature was placed 5 minutes, and the protein nucleic acid mixture is separated fully.
3. add the ratio adding chloroform of 200 μ l chloroforms with every 1ml Buffer RLM, build the pipe lid, thermal agitation 15 seconds, room temperature was placed 5 minutes.
4.4 ℃ 12, centrifugal 15 minutes of 000rpm, sample is divided into three layers: red organic phase, middle layer and colourless water move on to the colourless water in upper strata in the new centrifuge tube.
5. the dehydrated alcohol that adds 1/3 times of volume in the solution that obtains to step 4, mixing, the adsorption column RM(Spin Column RM that changes the solution that obtains and precipitation over to the collection tube of having packed into (Collection Tube2ml) together) in.If once complete soln can not be added adsorption column, please change over to several times.Centrifugal 30 seconds of 12,000rpm discards adsorption column RM after centrifugal, keeps effluent liquid.
6. the dehydrated alcohol that adds 2/3 times of volume in the solution that obtains to step 5, mixing.
7. will go up the adsorption column RS(Spin Column RS that step gained solution and precipitation change the collection tube of having packed into (Collection Tube2ml) together over to) in.If once complete soln can not be added adsorption column, please change over to several times.Centrifugal 30 seconds of 12,000rpm outwells waste liquid in the collection tube, adsorption column RS is relay reclaim in the collector.
8. add 700 μ l Buffer RWT(preoperation inspections in the adsorption column RS and whether add dehydrated alcohol), room temperature 12, centrifugal 30 seconds of 000rpm outwells the waste liquid in the collection tube, adsorption column RS is relay reclaim in the collector.
9. add 500 μ l Buffer RW2(preoperation inspections in the adsorption column RS and whether add dehydrated alcohol), room temperature 12, centrifugal 30 seconds of 000rpm outwells the waste liquid in the collection tube, adsorption column RS is relay reclaim in the collector.
10. repeating step 9.
11.12 centrifugal 1 minute of 000rpm outwells the waste liquid in the collection tube.Adsorption column RS is placed room temperature number minute, thoroughly to dry.Attention: the purpose of this step is that ethanol residual among the adsorption column RS is removed, residual can the influence follow-up enzymatic reaction of ethanol (enzyme is cut, PCR etc.).
12. adsorption column RS is placed new no RNA enzyme (RNase-free) centrifuge tube (Collection Tube1.5ml), the water that does not contain the RNA enzyme (RNase-Free Water) that adds 30 μ l to the adsorption column middle part, room temperature was placed 1 minute, 12, centrifugal 1 minute of 000rpm, collect RNA solution, the RNA solution that obtains is kept at-70 ℃, prevents degraded.
13. use microplate reader to detect gained RNA concentration, it is as shown in table 1 to detect gained Micro RNA concentration:
Table 1Micro RNA concentration
| 260/280 | ng/μL | Sample |
| 1.077 | -0.207 | ddH2O |
| 2.015 | 104.403 | Cal-27 |
| 1.897 | 79.606 | Hep-2 |
| 1.904 | 99.915 | LOVO |
| 2.097 | 84.689 | MCF-7 |
Processing and the reverse transcription of embodiment 4Micro RNA sample
1. primer oligo1,2 or 3 carries out annealing reaction
Above-mentioned three kinds of synthetic good primer oligo1, oligo2 with loop-stem structure and oligo3 are diluted to 50 μ M/ μ l respectively, prepare 50 μ l annealing reaction mixed solutions.Component in the mixed solution is: every kind of primer (Oligo1, oligo2 and oligo3) with loop-stem structure adds 10 μ l respectively, adds 5 * annealing buffer of 10 μ l again, and the ddH of 10 μ l
2O.
Above-mentioned mixing solutions is carried out annealing reaction at the PCR instrument, and response procedures is:
(1) 95 ℃ of reaction 2min, (2) descended 0.1 ℃ in per 5～10 seconds, were down to 35 ℃～15 ℃, (3) 4 ℃ of preservations.With the Oligo1 after the anneal, Oligo2 and Oligo3 mixing solutions, dilute 5～15 times, standby.
2.Micro RNA sample tailing is handled
With PCR pipe and the mark of tweezers gripping 200 μ l, add the Micro RNA sample of 1 μ g embodiment, 3 preparation gained, add NEB E.coli poly(A) polysaccharase 0.2 μ l, 10 * polymerase buffer, 1 μ l, 10mMATP1 μ l, moisturizing to 20 μ l, mixing is centrifugal.With 37 ℃ of reactions of this mixed solution 15min, namely get the Micro RNA sample that has added the PolyA tail, the concentration of described sample sees Table 1.
3. the Micro RNA sample behind the tailing is annealed with primer oligo1～3 and is connected
Other gets new PCR pipe, and every pipe adds Oligo1～3 mixing solutionss of 7.5 μ l above-mentioned steps, 1 annealing back gained, and the above-mentioned gained of 4 μ l adds the Micro RNA sample of PolyA tail, 1.25 μ l dNTP, moisturizing to 16.5 μ l is centrifugal behind the mixing.The gained mixed solution is carried out annealing reaction at the PCR instrument, and response procedures is: 70 ℃, reaction 10min namely gets the Micro RNA sample after the processing, with 4 ℃ of preservations of gained sample.
4. gained is added the Micro RNA sample reverse transcription of PolyA tail
Every pipe in the gained Micro RNA sample is added 5.25 μ l reverse transcription systems carry out reverse transcription after centrifugal, component such as the table 2 of described reverse transcription system show:
Table 2 reverse transcription system component
| Reagent | Add-on |
| 5 * Primer Script damping fluid | 4μl |
| Primer?Script | 0.5μl |
| The M-MLV-RT ThermoScript II | 0.75μl |
| Total amount | 5.25μl |
[0116]The program of described reverse transcription is: (1) 30 ℃ of reaction 13min; (2) 42 ℃ of reaction 55min; (3) 50 ℃ of reaction 12min; (4) 95 ℃ of reaction 15min; (5) with gained cDNA sample in 4 ℃ of preservations.
Utilize microplate reader to detect gained cDNA sample concentration, sample concentration is as shown in table 3:
Table 3cDNA sample concentration
| 260/280 | ng/μL | Sample |
| 1.043 | -0.207 | ddH2O |
| 1.869 | 1066.587 | Cal-27 |
| 1.995 | 998.246 | Hep-2 |
| 1.934 | 1129.912 | LOVO |
| 1.898 | 964.129 | MCF-7 |
Real-time quantitative PCR (Realtime-PCR) reaction system component is as shown in table 4:
Table 4 real-time quantitative PCR reaction system
The program of described Real time PCR reaction is:
(1) pre-sex change is 95 ℃, 1 minute; (2) sex change is 95 ℃, 5 seconds; (3) annealing is extended 60 ℃, 20 seconds; Step (2)～(3) are carried out 40 circulations altogether, read light absorption value in the extension stage at every turn.
Begin to warm to 95 ℃ from 65 ℃ after the PCR EP (end of program), per second 0.5 degree that rises obtains melting curve.Described Real time PCR reaction result is shown in table 5 and table 6:
Table 5Real time pcr amplification result (2
-Δ Δ CtMethod)
| Group | MiR365a | U6 | ΔCt | - |
2 -ΔΔCt |
| Cal-27 |
24.99 | 20.39 | 4.633333 | -0.03 | 0.98 |
| Cal-27 |
24.95 | 20.30 | 4.593333 | 0.01 | 1.01 |
| Cal-27 repeats 3 | 24.94 | 20.38 | 4.583333 | 0.02 | 1.01 |
| Hep2 repeats 1 | 29.15 | 25.18 | 8.793333 | -4.19 | 0.05 |
| Hep2 repeats 2 | 29.41 | 25.33 | 9.053333 | -4.45 | 0.05 |
| Hep2 repeats 3 | 29.37 | 25.24 | 9.013333 | -4.41 | 0.05 |
| MCF-7 |
26.72 | 19.42 | 6.363333 | -1.76 | 0.30 |
| MCF-7 |
26.65 | 19.42 | 6.293333 | -1.69 | 0.31 |
| MCF-7 repeats 3 | 26.64 | 19.52 | 6.283333 | -1.68 | 0.31 |
| LOVO repeats 1 | 31.35 | 28.08 | 10.99333 | -6.39 | 0.01 |
| LOVO repeats 2 | 31.55 | 28.29 | 11.19333 | -6.59 | 0.01 |
| LOVO repeats 3 | 31.38 | 28.01 | 11.02333 | -6.42 | 0.01 |
Table 6Real time pcr amplification is statistical data analysis as a result
| Sample | Mean value | Standard deviation |
| Cal-27 | 1.00 | 0.018258333 |
| Hep2 | 0.05 | 0.004887644 |
| MCF-7 | 0.31 | 0.009160718 |
| LOVO | 0.01 | 0.000829451 |
Find through fluorescence quantitative PCR detection, carry out reverse transcription by this counter-rotating system after, four kinds of used cell strains all can be finished small segment mRNA(U6,106nt) and micro RNA(MiR-365a, counter-rotating 21nt) and amplification.From Fig. 1 (A～D), Fig. 2 (A～D), Fig. 3 (A～D) and Fig. 4 (and the A～D) in what amplification curve of melting curve of each clone as can be seen: the fragment that amplifies is single fragment, there is not bimodal pollution, further specifies to present truly by reverse transcription system of the present invention and comprise small fragment RNA (about 100nt) and the Micro RNA basal expression level in each clone.Data in the table 5 are passed through 2
-Δ Δ CtMethod is analyzed, and draws Cal-27, the basal expression amount of Micro RNA365a in Hep2, MCF-7 and the LOVO cell strain, and its result is as shown in Figure 5.
With respect to the present common primer with neck ring structure, this reverse transcription system can be by reverse transcription reaction simply and efficiently, finish the reverse transcription that comprises confidential reference items and Micro RNA to be checked, greatly reduce the substep systematic error that the small segment mRNA of Micro RNA and confidential reference items produces of reversing, and saved the time.
The comparative example 1
The Micro RNA sample that adopts example 3 to handle gained reverses, and uses the Bulge-Loop of the rich sharp bio tech ltd in Guangzhou
TMMicro RNA365a qRT-PCR Primer (article No. miRQ0000710-1-2) carries out reverse transcription.
1, gets 5mM RT primer working fluid 1 μ l, add water (the RNase-free H that 79 μ l do not contain the RNA enzyme
2O), be configured to 62.5nM reverse transcription primer working fluid.
2, adopt 25 μ l reverse transcription reaction systems, Micro RNA reverse transcription the first step system is as shown in table 7:
Table 7Micro RNA reverse transcription the first step system
| Reagent | 25 μ l systems | Final concentration |
| RNA template (1 μ g) | 1μl | ? |
| Oligo?dT(20nM) | 6.5μl | 5nM |
| The water that does not contain the RNA enzyme | To 11 μ l | ? |
U6 gene reverse transcription the first step system is as shown in table 8:
Table 8U6 gene reverse transcription the first step system
| Reagent | 25 μ l systems | Final concentration |
| RNA template (1 μ g) | 1μl | ? |
| RT primer working fluid (62.5nM) | 2μl | 5nM |
| The water that does not contain the RNA enzyme | To 11 μ l | ? |
Behind the above system mixing, instantaneous centrifugal, to place 10 minutes for 70 ℃, ice bath 2 minutes adds following reagent again and carries out reverse transcription (RT) reaction, the RT response procedures of standard: 42 ℃ of 60min, 70 ℃ of 10min.
Reverse transcription second step system is as shown in table 9:
Table 9 reverse transcription second step system
Please immediately the cDNA product is taken out after RT reaction finishes, put cooled on ice fast, follow-up institute all carries out on ice in steps, please not with the cDNA product at will from removing on ice.
2) Micro RNA qPCR reaction
I.SYBR Green Mix (available from Bio-Rad): comprise Taq enzyme, dNTP mix, PCR Buffer, SYBR Green, the Micro RNA reaction system of qRT-PCR is as shown in table 10:
Table 10Bulge-Loop
TMThe Micro RNA reaction system of qRT-PCR
The internal control gene U6 reaction system of qRT-PCR is as shown in table 11:
Table 11Bulge-Loop
TMThe internal control gene U6 reaction system of qRT-PCR:
| Reactive component | The 20ul system | Final concentration |
| SYBR Green mixture | 9μl | 1X |
| The RT product | 2μl | ? |
| U6 forward primer (5 μ M) | 2μl | 500nM |
| U6 reverse primer (5 μ M) | 2μl | 500nM |
| The water that does not contain the RNA enzyme | To 20 μ l | The forms bottom |
Ii. use the three-step approach of standard to detect (Bio-Rad CFX96), program is as follows:
(1) pre-sex change is 95 ℃, 1 minute; (2) sex change is 95 ℃, 5S; (3) annealing is extended 60 ℃, 20S; Step (2)～(3) are carried out 40 circulations altogether, read light absorption value in the extension stage at every turn.
Begin to warm to 95 ℃ from 65 ℃ after the PCR EP (end of program), per second 0.5 degree that rises obtains melting curve.Described Realtime PCR reaction result is shown in table 12 and table 13:
Table 12RealtimePCR amplification (2-Δ Δ Ct method)
Data in the table 12 are analyzed by 2-Δ Δ Ct method, drawn Cal-27, the basal expression amount of Micro RNA365 in Hep2, MCF-7 and the LOVO cell strain, its result is as shown in Figure 6.
Result after the gained qRT-PCR data statistics is as shown in table 13:
Table 13RealtimePCR amplification statistical data analysis
Comparative example's 1 gained data are carried out variance analysis with gained qRT-PCR result of the present invention, and its result is as shown in table 14:
Table 14 comparing embodiment and differences of the present invention
The Micro RNA sample that the employed Micro RNA of comparative example and reverse transcription of the present invention is same batch of preparation has adopted 1 μ gMicro RNA sample to carry out reverse transcription as template equally.Result with table 5 relatively shows the back with table 12, adopts Bulge-Loop
TMBehind the Micro RNA365a qRT-PCRPrimer, the expression amount of Micro RNA365a obviously descends, and the CT value uses the CT value of Micro RNA365a of neck ring structure primer reverse transcription gained of the present invention big, and Δ CT value also becomes bigger than normal.The above results shows: adopt the existing Micro RNA365a Micro RNA365a with primer reverse transcription gained of neck ring structure more provided by the invention with primer reverse transcription gained of neck ring structure to measure low a lot.And use the U6 gene of Oligo dT reverse transcription gained equally, similar substantially to the reverse U6 gene that obtains of the present invention.Though finally express both unanimities of trend, the present invention can embody actual Micro rna expression situation better.The present invention simultaneously need not Micro RNA and internal control gene are reversed step by step, has improved Micro RNA and has detected the working efficiency of operating.
Should be understood that after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (9)
1. a Micro rna expression detection method is characterized in that, described Micro rna expression detection method may further comprise the steps:
(1) the Micro RNA of extraction sample connects the PolyA tail with gained Micro RNA;
(2) carry out anneal after three primers with loop-stem structure are mixed, the sequence of described primer with loop-stem structure is respectively shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ IDNO:3 in the sequence table;
(3) the Micro RNA that step (1) gained is connected with Poly A tail is connected with the primer annealing that step (2) gained has loop-stem structure;
(4) be that the template reverse transcription obtains cDNA with step (3) gained Micro RNA;
(5) be template with step (4) gained cDNA, add the primer of amplification target Micro RNA, detect the expression of target Micro RNA by fluorescence quantifying PCR method.
2. Micro rna expression detection method as claimed in claim 1 is characterized in that, the program of the described anneal of step (2) is: (1) 95 ℃ of reaction 2min, (2) descended 0.1 ℃ in per 5～10 seconds, were down to 35 ℃～15 ℃, (3) 4 ℃ of preservations.
3. Micro rna expression detection method as claimed in claim 1 is characterized in that, the program that the described annealing of step (3) connects is: 60～70 ℃, and reaction 5～10min.
4. Micro rna expression detection method as claimed in claim 1 is characterized in that, the program of the described reverse transcription of step (4) is: (1) 30 ℃ was reacted 10～15 minutes; (2) 42 ℃ were reacted 50～60 minutes; (3) 50 ℃ were reacted 10～15 minutes; (4) 95 ℃ were reacted 10～15 minutes; (5) with gained cDNA sample in 4 ℃ of preservations.
5. Micro rna expression detection method as claimed in claim 1, it is characterized in that, the primer of the described amplification target of step (5) Micro RNA is: the general reverse primer of Micro RNA and MicroRNA detect forward primer, and described Micro RNA detects the sequence of forward primer shown in SEQ ID NO:5.
6. the primer with loop-stem structure is characterized in that, the sequence of described primer with loop-stem structure is shown in SEQ ID NO:1, SEQ ID NO:2 in the sequence table or SEQ ID NO:3.
7. Micro RNA detection by quantitative test kit, it is characterized in that, this Micro RNA detection by quantitative test kit comprises: SYBR Green I fluorescence dye, PCR damping fluid, dNTPs solution, archaeal dna polymerase, aseptic double-distilled water, the primer with neck ring structure, general reverse primer and Micro RNA detect forward primer, and the sequence of wherein said primer with neck ring structure is respectively shown in SEQ ID NO:1, SEQ ID NO:2 in the sequence table and SEQ ID NO:3.
8. Micro RNA detection by quantitative test kit as claimed in claim 7 is characterized in that, described Micro RNA detects the sequence of forward primer shown in SEQ ID NO:5 in the sequence table.
9. the application of each described Micro RNA detection by quantitative test kit of claim 7～8 in detecting the Micro rna expression.
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