CN105039337A

CN105039337A - 5'RACE RNA adapter sequence and kit for amplifying terminal of miRNA sheared target gene cDNA 5'

Info

Publication number: CN105039337A
Application number: CN201510546723.6A
Authority: CN
Inventors: 张冉; 韦朝领; 张骁; 张菲; 师聪
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2015-08-31
Filing date: 2015-08-31
Publication date: 2015-11-11
Anticipated expiration: 2035-08-31
Also published as: CN105039337B

Abstract

The invention provides an efficient 5'RACE RNA adapter sequence which is shown as Seq ID No.1 and further provides a kit for amplifying a terminal of a miRNA sheared target gene cDNA 5' containing the adapter sequence. The 5'RACE RNA adapter sequence in the market is optimized, so that the possibility of a dipolymer and a hairpin structure of the primer of the 5'RACE RNA adapter sequence is reduced to a great extent, the terminal sequence of the miRNA sheared target gene cDNA 5' is amplified in a higher-sensitivity and higher-specificity mode, and the break locus of the miRNA target gene is efficiently verified. Compared with like kit products in the market, the kit is lower in cost and higher in sensitivity and accuracy.

Description

The test kit of 5 ' RACE RNA joint sequence and the target gene cDNA 5 ' end after shearing for the miRNA that increases

Technical field

The present invention relates to biology field, specifically, relate to the test kit of a kind of efficient RNA joint sequence for 5 ' RACE and the target gene cDNA5 ' end after shearing for the miRNA that increases, described RNA joint sequence can increase miRNA shear after target gene cDNA5 ' end sequence, thus carry out miRNA target gene checking.

Background technology

CDNA end rapid amplifying technology (rapidamplificationofcDNAends, RACE) being the effective ways of a kind of PCR-based 5' and 3 ' end of rapid amplifying cDNA from low-abundance transcript, is the best approach according to the known partial sequence amplification unknown fragment of gene.It is exactly combination by reverse transcription and PCR that RACE technology is put it briefly, the technology of amplification gene unknown fragment.Utilize the known array that gene coding region 3 ' is held, the technology obtaining 5 ' end unknown nucleotide sequence is called 5 '-RACE, is the most common technique of amplification 5 ' end unknown gene complete encoding sequence at present.

5 '-RACE ultimate principle: first obtain the RNA sample comprising goal gene mRNA, and utilize the primer of gene specific to carry out reverse transcription, obtains the corresponding cDNA of goal gene; Specific sequence is added at the 3 ' end (corresponding mRNA sequence 5 ' end) of cDNA, usually by this section, the particular sequence be added on cDNA is called joint (adaptor), synthesize the primer matched with adaptor sequence simultaneously, be called anchor primer; Then utilize gene known array to design gene-specific primer, match with anchor primer and carry out pcr amplification, obtain the fragment of unknown portions; Again by the sequencing fragment obtained that increases, the sequence of goal gene unknown portions can be obtained.

3 large classes can be divided at present: terminal enzyme (DNA) method (terminaldeoxynucleotidyltransferasemethod, TdT method), joint connection method (adaptorligation) and oligonucleotide cap method (oligocappingmethod) by the addition means of joint sequence.

Oligonucleotide cap method is also known as RLM-RACE.Eukaryote mRNA has a 3 ' tail and a 5 ' cap sequence, and the cap characteristic that this technology utilizes 5 ' to hold just carries out enrichment and amplification to full-length molecule, can locate transcription initiation site more accurately.Test kit at present based on the method has the producers such as TaKaRa, Clontech and Ambion to have production.

Summary of the invention

The object of this invention is to provide 5 ' RACERNA joint sequence of the 5 ' end of the target gene cDNA after a kind of amplification miRNA shearing of more highly sensitive, more high specific.

Another object of the present invention is to provide the test kit for the target gene cDNA5 ' end after miRNA shearing of increasing containing above-mentioned 5 ' RACERNA joint sequence.

In order to realize the object of the invention, the efficient RNA joint sequence for 5 ' RACE provided by the invention, described RNA joint sequence is: 5'-GCGUGACCACUAGUCACGUCUGAGUUCGUCGCCAUUCCACAAA-3'(SeqIDN is o.1).

The present invention also provides the test kit for the target gene cDNA5 ' end after miRNA shearing of increasing containing above-mentioned joint sequence.

Aforesaid test kit also comprises 5 ' RACE Outside primer for nest-type PRC and 5 ' RACE inner primer, and primer sequence is as follows:

5 ' RACE Outside primer: 5'-ACTAGTCACGTCTGAGTTCG-3'(SeqIDNo.2)

5 ' RACE inner primer: 5'-CGCGGATCCACGTCTGAGTTCGTCGCCATTCCAC-3'(SeqIDNo.3)

Aforesaid test kit also comprises T4RNA ligase enzyme, RNA ligase damping fluid, RNase inhibitor, ThermoScript II M-MLV, M-MLV damping fluid, dNTPs, Random9mers, RNaseFreedH ₂o, Taq DNA polymerase, Mg ²⁺, at least one in PCR reaction buffer etc.

The present invention also provides the method for the target gene cDNA5 ' end sequence after utilizing 5 ' RACE amplification miRNA to shear, and comprises the following steps:

1) Auele Specific Primer GSP2 inside the upstream outer Auele Specific Primer GSP1 of nest-type PRC and upstream is designed for according to the cDNA sequence that goal gene is known;

2) with T4RNA ligase enzyme, 5 ' RACE joint is connected on the said target mrna after miRNA shearing;

3) with step 4) said target mrna be template, Random9mers is reverse transcription primer, with ThermoScript II M-MLV carry out reverse transcription synthesis cDNA;

4) with step 5) cDNA be template, carry out nest-type PRC reaction, carry out first round PCR reaction with upstream outer Auele Specific Primer GSP1 and 5 ' RACE Outside primer, then with Auele Specific Primer GSP2 and 5 ' RACE inner primer inside upstream carry out second take turns PCR reaction;

5) PCR primer is analyzed.

Wherein, step 4) described in the sequence of 5 ' RACE joint as follows: 5'-GCGUGACCACUAGUCACGUCUGAGUUCGUCGCCAUUCCACAAA-3'(SeqIDN is o.1).

Step 4) described in the sequence of 5 ' RACE Outside primer and 5 ' RACE inner primer as follows respectively:

5 ' RACE Outside primer: 5'-ACTAGTCACGTCTGAGTTCG-3'(SeqIDNo.2)

5 ' RACE inner primer: 5'-CGCGGATCCACGTCTGAGTTCGTCGCCATTCCAC-3'(SeqIDNo.3)

Aforesaid method, step 1) in design of primers principle as follows:

1. primer length is 20-28nt;

2. in primer, GC content is 45%-55%, GC, AT is evenly distributed, and avoids local to be rich in GC or AT;

3. primer does not form secondary structure, does not form complex construction with 5 ' RACE Outside primer and 5 ' RACE inner primer;

4. 3-4 the base that primer 3 ' is held does not form complementary sequence with pairing primer.

Aforesaid method, step 2) concrete operations as follows:

A) the said target mrna 5 μ l after reaction solution I:miRNA shearing is prepared, 15 μM of 5 ' RACE joint 1 μ l, RNaseFreedH ₂o4 μ l;

B) reaction solution I places 2 minutes in 65 DEG C of insulations on ice after 5 minutes, then in reaction solution I, 40U/ μ lRNase inhibitor 1 μ l is added, 5 × RNA ligase damping fluid 8 μ l, 40%PEG600020 μ l, 40U/ μ lT4RNA ligase enzyme 1 μ l, obtains reaction solution II:

C) reaction solution II is reacted 1 hour in 16 DEG C;

D) in reaction solution c), add the 3MCH of 20 μ lpH5.2 ₃cOONa, the RNaseFreedH of 140 μ l ₂mix after O;

E) in reaction solution d), phenol/chloroform/primary isoamyl alcohol that 200 μ l volume ratios are 25:24:1 is added, centrifugal 5 minutes of 13000g room temperature after mixing, by upper water phase transition in new Microtube;

F) in reaction solution e), the chloroform of 200 μ l is added, centrifugal 5 minutes of 13000g room temperature after mixing, by upper water phase transition in new Microtube;

G) mix add the NACarrier of 2 μ l in reaction solution f) after;

H) in reaction solution g), the Virahol of 200 μ l is added, cooled on ice 10 minutes after mixing;

I) 13000g4 DEG C centrifugal 20 minutes, abandon supernatant; Add the 70% cold ethanol rinse of 500 μ l, 13000g4 DEG C centrifugal 5 minutes, dry after abandoning supernatant;

J) RNaseFreedH of 6 μ l is added ₂o dissolution precipitation, obtains LigatedRNA.

Aforesaid method, step 3) concrete operations as follows:

K) inverse transcription reaction liquid is prepared: LigatedRNA6 μ l, 50 μMs of Random9mers0.5 μ l, 5 × M-MLV damping fluid 2 μ l, 10mMdNTPs1 μ l, 40U/ μ lRNase inhibitor 0.25 μ l, 200U/ μ l ThermoScript II M-MLV0.25 μ l;

L) carry out reverse transcription reaction by following condition: 30 DEG C 10 minutes; 42 DEG C 1 hour; 70 DEG C 15 minutes.

Aforesaid method, step 4) concrete operations as follows:

M) first round PCR reaction solution is prepared: inverse transcription reaction liquid 2-5 μ l, 1 × cDNA dilution buffer 5-8 μ l, 10 × PCR reaction buffer 4 μ l, 25mMMgCl ₂3 μ l, 5U/ μ lTaqDNA polysaccharase 0.25 μ l, 10 μMs of upstream outer Auele Specific Primer GSP12 μ l, 10 μM of 5 ' RACE Outside primer 2 μ l, uses dH ₂o complements to 50 μ l;

N) carry out first round PCR reaction by following condition: 94 DEG C 3 minutes; 94 DEG C 30 seconds, 58 DEG C 30 seconds; 72 DEG C 1 minute, totally 28 circulations; 72 DEG C 10 minutes;

O) prepare second and take turns PCR reaction solution: first round PCR reaction solution 1 μ l, 10 × PCR reaction buffer 5 μ l, 25mMMgCl ₂5 μ l, 2.5mMdNTPs8 μ l, 5U/ μ lTaqDNA polysaccharase 0.5 μ l, Auele Specific Primer GSP22 μ l inside 10 μMs of upstreams, 10 μM of 5 ' RACE inner primer 2 μ l, uses dH ₂o complements to 50 μ l;

P) by following condition carry out second take turns PCR reaction: 94 DEG C 3 minutes; 94 DEG C 30 seconds, 60 DEG C 30 seconds; 72 DEG C 1 minute, totally 28 circulations; 72 DEG C 10 minutes.

The present invention further provides the described efficient RNA joint sequence for 5 ' RACE or the application of described test kit in research miRNA and target gene interact.

The present invention has the following advantages:

(1) the present invention is by being optimized the efficient RNA joint sequence of 5 ' RACE, make its primer largely decrease the possibility of dimer and hairpin structure, can the amplification miRNA of more highly sensitive, more high specific shear after the 5 ' end sequence of target gene cDNA.

(2) the present invention can the broken site of high specific, efficiently checking miRNA target gene, and compared with reagent kit product similar on market, susceptibility is higher, and accuracy is stronger.

Accompanying drawing explanation

Fig. 1 is the amplification schematic diagram utilizing the test kit of the embodiment of the present invention 1 to carry out 5 ' RACE.

Fig. 2 is forming process and the mode of action schematic diagram of miRNA in plant.

Fig. 3 utilizes the interactional agarose gel electrophoresis figure of 5 ' RACERNA joint sequence checking miR396d and target gene isotig01346 in the embodiment of the present invention 3.

Fig. 4 utilizes the interactional agarose gel electrophoresis figure of 5 ' RACERNA joint sequence checking miR408 and target gene 24872_gi400019881 in the embodiment of the present invention 3.

Fig. 5 utilizes the interactional agarose gel electrophoresis figure of 5 ' RACERNA joint sequence checking miR399 and target gene 76159_gi319841213 in the embodiment of the present invention 3.

Fig. 6 utilizes the interactional agarose gel electrophoresis figure of 5 ' RACERNA joint sequence checking miR156 and target gene isotig01056 in the embodiment of the present invention 3.

Fig. 7 is miRNA396d and its target gene isotig01346 interaction schematic diagram in the embodiment of the present invention 3.

In Fig. 3-Fig. 6, swimming lane 1 is the experimental result of the joint sequence in TaKaRa company test kit, and swimming lane 2 is the DNAMarker (D2000Plus) of 2000bp, and swimming lane 3 is the experimental result of 5 ' RACERNA joint sequence of the present invention.

Embodiment

Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.

Embodiment 1 is for 5 ' RACE test kit of the target gene cDNA5 ' end after miRNA shearing of increasing

5 ' RACE the test kit for the target gene cDNA5 ' end after miRNA shearing of increasing that the present embodiment provides is composed as follows:

Reaction (10 secondary amounts) for RNA process: 5 ' RACEAdaptor (15 μMs) 10 μ l, T4RNA ligase enzyme (40U/ μ l) 10 μ l, 5 × RNA ligase damping fluid 80 μ l, 40%PEG6000200 μ l, 3MCH ₃cOONa (pH5.2) 400 μ l, NACarrier40 μ l, RNase inhibitor (40U/ μ l) 32.5 μ l, ThermoScript II M-MLV (RNaseH ^-) (200U/ μ l) 2.5 μ l, 5 × M-MLV damping fluid 20 μ l, dNTPMixture (each 10mM) 10 μ l, Random9mers (50 μMs) 5 μ l, RNaseFreedH ₂o2 × 1ml.

For 5 ' RACEPCR reaction (50 secondary amounts): 1 × cDNA dilution buffer II400 μ l5 ' RACEOuterPrimer (10 μMs) 100 μ l, 5 ' RACEInnerPrimer (10 μMs) 100 μ l.

Wherein, 5 ' RACEAdaptor and primer sequence as follows:

5′RACEAdaptor：5'-GCGUGACCACUAGUCACGUCUGAGUUCGUCGCCAUUCCACAAA-3'(SeqIDNo.1)

5′RACEOuterPrimer：5'-ACTAGTCACGTCTGAGTTCG-3'(SeqIDNo.2)

5′RACEInnerPrimer：5'-CGCGGATCCACGTCTGAGTTCGTCGCCATTCCAC-3'(SeqIDNo.3)

In addition, the reagent that needs prepare is:

(1) Auele Specific Primer GSP2 inside the upstream outer Auele Specific Primer GSP1 of nest-type PRC and upstream is designed for according to the cDNA sequence that goal gene is known.Design of primers principle is as follows: 1. primer length is 20-28nt; 2. in primer, GC content is 45%-55%, GC, AT is evenly distributed, and avoids local to be rich in GC or AT; 3. primer does not form secondary structure, does not form complex construction with 5 ' RACE Outside primer and 5 ' RACE inner primer; 4. 3-4 the base that primer 3 ' is held does not form complementary sequence with pairing primer.

(2) PCR archaeal dna polymerase.The TaKaRaLA that recommendation amplification capability is good or the high PrimeSTAR of fidelity performance (TaKaRaCode:DRR02AM) ^tMhSDNAPolymerase (TaKaRaCode:DR010).

(3) PCR primer cloning kit (as: carrier T etc.).

(4) phenol/chloroform/primary isoamyl alcohol (25/24/1).

(5) chloroform, Virahol, 100% pre-cooled ethanol, 70% pre-cooled ethanol.

This test kit Ying Yu-20 DEG C preservation.

Test kit is utilized to carry out the amplification principle of 5 ' RACE as shown in Figure 1.Operation steps is as follows:

1,5 ' RACEAdaptor is connected on the said target mrna on the mRNA after cap or after miRNA shears with T4RNA ligase enzyme;

2, with the mRNA of step/3 for template, Random9mers is reverse transcription primer, with ThermoScript II M-MLV carry out reverse transcription synthesis cDNA;

3, with the cDNA of step/4 for template, carry out nest-type PRC reaction, carry out first round PCR reaction with upstream outer Auele Specific Primer GSP1 and 5 ' RACEOuterPrimer, then with Auele Specific Primer GSP2 and 5 ' RACEInnerPrimer inside upstream carry out second take turns PCR reaction;

4, PCR primer is analyzed.

The method of the target gene cDNA5 ' end sequence after embodiment 2 utilizes 5 ' RACE amplification miRNA to shear

The test kit of embodiment 1 is utilized to carry out 5 ' RACE to laboratory sample.

1, the connection of 5 ' RACEAdaptor.

1. first following solutions is prepared: the said target mrna 5 μ l after miRNA shears, 5 ' RACEAdaptor (15 μMs) 1 μ l, RNaseFreedH ₂o4 μ l.

2. 65 DEG C of insulations placed 2 minutes on ice after 5 minutes, then added following reagent: RNase inhibitor (40U/ μ l) 1 μ l, 5 × RNA ligase damping fluid 8 μ l, 40%PEG600020 μ l, T4RNA ligase enzyme (40U/ μ l) 1 μ l.

3. 16 DEG C are reacted 1 hour.

4. in above-mentioned reaction solution, add the 3MCH of 20 μ l ₃cOONa (pH5.2), the RNaseFreedH of 140 μ l ₂after O, fully mix.

5. phenol/chloroform/the primary isoamyl alcohol (25:24:1) of 200 μ l is added, fully centrifugal 5 minutes of rear 13, the 000 × g room temperatures of mixing, by upper water phase transition in new Microtube.

6. the chloroform of 200 μ l is added, fully centrifugal 5 minutes of rear 13, the 000 × g room temperatures of mixing, by upper water phase transition in new Microtube.

7. Homogeneous phase mixing after the NACarrier of 2 μ l is added.

8. the Virahol of 200 μ l is added, fully after mixing, cooled on ice 10 minutes.

9. 13,000 × g4 DEG C centrifugal 20 minutes, abandon supernatant.Add the 70% cold ethanol (RNaseFreedH of 500 μ l ₂o prepares) rinsing, 13,000 × g4 DEG C are centrifugal 5 minutes, dry after abandoning supernatant.

10. the RNaseFreedH of 6 μ l is added ₂o dissolution precipitation, obtains LigatedRNA.

2, reverse transcription reaction.

1. by following component preparation inverse transcription reaction liquid: LigatedRNA6 μ l, Random9mers (50 μMs) 0.5 μ l, 5 × M-MLV damping fluid 2 μ l, dNTP (each 10mM) 1 μ l, RNase inhibitor (40U/ μ l) 0.25 μ l, ThermoScript II M-MLV (RNaseH ^-) (200U/ μ l) 0.25 μ l, cumulative volume 10 μ l.

For the gene of three-dimensional arrangement complexity, first reverse transcription reaction should be carried out again by after LigatedRNA sex change.Concrete operations are: first the mixture of LigatedRNA and Random9mers after 10 minutes, is placed 2min in 70 DEG C of sex change on ice, then all the other reagent adding reverse transcription reaction carry out reverse transcription reaction.

2. reverse transcription reaction condition is as follows: 30 DEG C 10 minutes; 42 DEG C 1 hour; 70 DEG C 15 minutes.

3. reaction can carry out next step experiment after terminating, or reaction solution is stored in-20 DEG C.

3, first round PCR reacts: OuterPCR reacts.

1. by following component preparation OuterPCR reaction solution: the inverse transcription reaction liquid X μ l of above-mentioned 4,1 × cDNA dilution buffer II10-X μ l, 10 × LAPCR reaction buffer II (Mg ²⁺free) 4 μ l, MgCl ₂(25mM) 3 μ l, TaKaRaLATaq (5U/ μ l) 0.25 μ l, upstream outer Auele Specific Primer GSP1 (10 μMs) 2 μ l, 5 ' RACEOuterPrimer (10 μMs) 2 μ l, uses dH ₂o complements to 50 μ l.

2. PCR reaction conditions is as follows: 94 DEG C 3 minutes; 94 DEG C 30 seconds, 58 DEG C 30 seconds; 72 DEG C of 1 minute/kb, totally 28 circulations; 72 DEG C 10 minutes.

Wherein, the recommendation amount X of inverse transcription reaction liquid is 2 μ l, and use range is 2-5 μ l.In addition, can suitably improve according to practical situation or reduce annealing temperature.Generally, the time that extends presses 1min/kb setting.The extension time can be suitably increased during Special Circumstances.

4, second PCR reaction is taken turns: InnerPCR reacts.

1. by following component preparation InnerPCR reaction solution: OuterPCR reaction solution 1 μ l, 10 × LAPCR reaction buffer II (Mg ²⁺free) 15 μ l, MgCl ₂(25mM) 5 μ l, dNTPMixture (each 2.5mM) 8 μ l, TaKaRaLATaq (5U/ μ l), Auele Specific Primer GSP2 (10 μMs) 2 μ l inside 0.5 μ l upstream, 5 ' RACEInnerPrimer (10 μMs) 2 μ l, uses dH ₂o complements to 50 μ l.

2. PCR reaction conditions is as follows: 94 DEG C 3 minutes; 94 DEG C 30 seconds, 60 DEG C 30 seconds; 72 DEG C of 1 minute/kb, totally 28 circulations; 72 DEG C 10 minutes.

Wherein, can suitably improve according to practical situation or reduce annealing temperature.Generally, the time that extends presses 1min/kb setting.The extension time can be suitably increased during Special Circumstances.In addition, depending on actual amplification situation, the circulation number of turns can be reduced to 20-30 circulation.

5, agarose gel electrophoresis.

After reaction terminates, get 5-10 μ lPCR reaction solution and carry out agarose gel electrophoresis, confirm pcr amplification product.If this PCR primer is tested after need being used for, by PCR primer in-20 DEG C of preservations.

6, sequencing analysis.

Following method can be adopted during order-checking:

1. PCR primer direct Sequencing.

Directly DNA sequencing is carried out by after the PCR primer purifying of 5 ' RACE amplification.The PCR primer of TaKaRa can be used to reclaim test kit (TaKaRaCode:DV807A) for the purifying of PCR primer and sepharose reclaims test kit (TaKaRaCode:DV805A; D301).If when PCR primer direct Sequencing cannot carry out, advise checking order after DNA fragmentation clone again.

2. PCR primer cloning and sequencing.

5 ' RACEPCR product checks order after can be cloned into suitable carrier.As used pMD18-TSimpleVector (TaKaRaCode:D103A) or pMD19-TSimpleVector (TaKaRaCode:D104A) etc.The two or more plasmid order-checking that Insert Fragment is longer should be chosen, because some gene exists different transcription initiation sites and shearing, connecting method during order-checking.

Embodiment 3 is for the application of RNA joint sequence in research miRNA and target gene interact of 5 ' RACE

MiRNA has very important adjusting function, they mainly participate in the regulation and control of level after genetic transcription, by causing the degraded of said target mrna (comparatively common in plant) with the specific base pairing of said target mrna or suppressing it to translate (comparatively common in animal), thus have impact on the expression (Fig. 2) of said target mrna.

According to the method for embodiment 2, be experiment material (said target mrna after shearing containing miRNA in the total serum IgE of extraction) with tea tree breed ' Shu Chazao ' total serum IgE, carry out following 5 ' RACE experiment respectively, to verify the interaction of miRNA and target gene.Experiment sets up TaKaRa company 5 '-FullRACEkitwithTAP test kit in contrast.

Wherein, the extraction of plant total serum IgE operates according to the plant total RNA extraction reagent box specification sheets of TIANGEN Biotech (Beijing) Co., Ltd..All miRNA and its target gene obtain by degraded group order-checking in tea tree below, then obtain accurate result through cloning and sequencing.

1, the target gene isotig01346 of miR396d is verified, increased the 5 ' RACE object fragment (SeqIDNo.4) of 284bp, its GSP1:5 '-ATCAATGAAATGGCGAAGTGG-3 ', GSP2:5 '-ATGTTGCTTTGGAATGGAGAATG-3 '.Experimental result is shown in Fig. 3.

2, the target gene 24872_gi400019881 of miR408 is verified, the 5 ' RACE object fragment (SeqIDNo.5) of the 173bp that increased, its GSP1:5 '-ATAGATGGGATGGGAGCA-3 ', GSP2:5 '-GCCTTGTTCACACTGACCAC-3 '.Experimental result is shown in Fig. 4.

3, the target gene 76159_gi319841213 of miR399 is verified, increased the 5 ' RACE object fragment (SeqIDNo.6) of 215bp, its GSP1:5 '-GCTTGATAGAATGTTACTGGC-3 ', GSP2:5 '-ATTACCAACACTCCATGTCTTCT-3 '.Experimental result is shown in Fig. 5.

4, the target gene isotig01056 of miR156 is verified, the 5 ' RACE object fragment (SeqIDNo.7) of the 153bp that increased, its GSP1:5 '-GTTAGGAAGGAAGTGACTGAAT-3 ', GSP2:5 '-TGCTCAGTCTGCCAATAGTCT-3 '.Experimental result is shown in Fig. 6.

As can be seen from Fig. 3-Fig. 6, adopt the RNA joint sequence for 5 ' RACE of the present invention to carry out miRNA and the interactional checking of target gene, efficiency is higher than the joint sequence in TaKaRa company 5 '-FullRACEkitwithTAP test kit.

Interact for miRNA396d and its target gene isotig01346, the schematic diagram of two-way interaction as shown in Figure 7.

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims

The efficient RNA joint sequence of 1.5 ' RACE, is characterized in that, described RNA joint sequence is: 5 '-GCGUGACCACUAGUCACGUCUGAGUUCGUCGCCAUUCCACAAA-3 '.
2. the test kit for the target gene cDNA5 ' end after miRNA shearing of increasing containing joint sequence described in claim 1.
3. test kit according to claim 2, is characterized in that, described test kit also comprises 5 ' RACE Outside primer for nest-type PRC and 5 ' RACE inner primer, and primer sequence is as follows:

5 ' RACE Outside primer: 5 '-ACTAGTCACGTCTGAGTTCG-3 '

5 ' RACE inner primer: 5 '-CGCGGATCCACGTCTGAGTTCGTCGCCATTCCAC-3 '.
4. the test kit according to Claims 2 or 3, it is characterized in that, described test kit also comprises T4RNA ligase enzyme, RNA ligase damping fluid, RNase inhibitor, ThermoScript II M-MLV, M-MLV damping fluid, dNTPs, Random9mers, RNaseFreedH ₂o, Taq DNA polymerase, Mg ²⁺, at least one in PCR reaction buffer.
5. the method for the target gene cDNA5 ' end sequence after utilizing 5 ' RACE amplification miRNA to shear, is characterized in that, comprise the following steps:

1) Auele Specific Primer GSP2 inside the upstream outer Auele Specific Primer GSP1 of nest-type PRC and upstream is designed for according to the cDNA sequence that goal gene is known;

2) with T4RNA ligase enzyme, 5 ' RACE joint is connected on the said target mrna after miRNA shearing;

3) with step 2) said target mrna be template, Random9mers is reverse transcription primer, with ThermoScript II M-MLV carry out reverse transcription synthesis cDNA;

4) with step 3) cDNA be template, carry out nest-type PRC reaction, carry out first round PCR reaction with upstream outer Auele Specific Primer GSP1 and 5 ' RACE Outside primer, then with Auele Specific Primer GSP2 and 5 ' RACE inner primer inside upstream carry out second take turns PCR reaction;

5) PCR primer is analyzed;

Wherein, step 2) described in the sequence of 5 ' RACE joint as follows: 5 '-GCGUGACCACUAGUCACGUCUGAGUUCGUCGCCAUUCCACAAA-3 ';

Step 4) described in the sequence of 5 ' RACE Outside primer and 5 ' RACE inner primer as follows respectively:

5 ' RACE Outside primer: 5 '-ACTAGTCACGTCTGAGTTCG-3 '

5 ' RACE inner primer: 5 '-CGCGGATCCACGTCTGAGTTCGTCGCCATTCCAC-3 '.
6. method according to claim 5, is characterized in that, step 1) in design of primers principle as follows:

1. primer length is 20-28nt;

2. in primer, GC content is 45%-55%, GC, AT is evenly distributed, and avoids local to be rich in GC or AT;

3. primer does not form secondary structure, does not form complex construction with 5 ' RACE Outside primer and 5 ' RACE inner primer;

4. 3-4 the base that primer 3 ' is held does not form complementary sequence with pairing primer.
7. method according to claim 5, is characterized in that, step 2) concrete operations as follows:

A) the said target mrna 5 μ l after reaction solution I:miRNA shearing is prepared, 15 μM of 5 ' RACE joint 1 μ l, RNaseFreedH ₂o4 μ l;

B) reaction solution I places 2 minutes in 65 DEG C of insulations on ice after 5 minutes, then in reaction solution I, 40U/ μ lRNase inhibitor 1 μ l is added, 5 × RNA ligase damping fluid 8 μ l, 40%PEG600020 μ l, 40U/ μ lT4RNA ligase enzyme 1 μ l, obtains reaction solution II:

C) reaction solution II is reacted 1 hour in 16 DEG C;

D) in reaction solution c), add the 3MCH of 20 μ lpH5.2 ₃cOONa, the RNaseFreedH of 140 μ l ₂mix after O;

E) in reaction solution d), phenol/chloroform/primary isoamyl alcohol that 200 μ l volume ratios are 25:24:1 is added, centrifugal 5 minutes of 13000g room temperature after mixing, by upper water phase transition in new Microtube;

F) in reaction solution e), the chloroform of 200 μ l is added, centrifugal 5 minutes of 13000g room temperature after mixing, by upper water phase transition in new Microtube;

G) mix add the NACarrier of 2 μ l in reaction solution f) after;

H) in reaction solution g), the Virahol of 200 μ l is added, cooled on ice 10 minutes after mixing;

I) 13000g4 DEG C centrifugal 20 minutes, abandon supernatant; Add the 70% cold ethanol rinse of 500 μ l, 13000g4 DEG C centrifugal 5 minutes, dry after abandoning supernatant;

J) RNaseFreedH of 6 μ l is added ₂o dissolution precipitation, obtains LigatedRNA.
8. method according to claim 7, is characterized in that, step 3) concrete operations as follows:

K) inverse transcription reaction liquid is prepared: LigatedRNA6 μ l, 50 μMs of Random9mers0.5 μ l, 5 × M-MLV damping fluid 2 μ l, 10mMdNTPs1 μ l, 40U/ μ lRNase inhibitor 0.25 μ l, 200U/ μ l ThermoScript II M-MLV0.25 μ l;

L) carry out reverse transcription reaction by following condition: 30 DEG C 10 minutes; 42 DEG C 1 hour; 70 DEG C 15 minutes.
9. method according to claim 8, is characterized in that, step 4) concrete operations as follows:

M) first round PCR reaction solution is prepared: inverse transcription reaction liquid 2-5 μ l, 1 × cDNA dilution buffer 5-8 μ l, 10 × PCR reaction buffer 4 μ l, 25mMMgCl ₂3 μ l, 5U/ μ lTaqDNA polysaccharase 0.25 μ l, 10 μMs of upstream outer Auele Specific Primer GSP12 μ l, 10 μM of 5 ' RACE Outside primer 2 μ l, uses dH ₂o complements to 50 μ l;

N) carry out first round PCR reaction by following condition: 94 DEG C 3 minutes; 94 DEG C 30 seconds, 58 DEG C 30 seconds; 72 DEG C 1 minute, totally 28 circulations; 72 DEG C 10 minutes;

O) prepare second and take turns PCR reaction solution: first round PCR reaction solution 1 μ l, 10 × PCR reaction buffer 5 μ l, 25mMMgCl ₂5 μ l, 2.5mMdNTPs8 μ l, 5U/ μ lTaqDNA polysaccharase 0.5 μ l, Auele Specific Primer GSP22 μ l inside 10 μMs of upstreams, 10 μM of 5 ' RACE inner primer 2 μ l, uses dH ₂o complements to 50 μ l;

P) by following condition carry out second take turns PCR reaction: 94 DEG C 3 minutes; 94 DEG C 30 seconds, 60 DEG C 30 seconds; 72 DEG C 1 minute, totally 28 circulations; 72 DEG C 10 minutes.
10. the application of test kit described in 5 ' RACERNA joint sequence described in claim 1 or any one of claim 2-4 in research miRNA and target gene interact.