CN104372083B - A kind of method for designing of the stem ring primer of reverse transcription miR-505 - Google Patents
A kind of method for designing of the stem ring primer of reverse transcription miR-505 Download PDFInfo
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Abstract
The present invention relates to the method for designing of the stem ring primer of a kind of reverse transcription miR 505, including: it is specific transcript with miRNA 505 3P gene, respectively by several gradients corresponding for the design of the quantity of stem's base pair of this reverse transcription stem ring primer and the base number rule of ring portion, through to the reverse transcription of miRNA 505 3P and real time PCR detection, finishing screen selects one for the optimal reverse transcription stem ring primer of efficiency during miRNA 505 3P gene specific reverse transcription.Present invention, avoiding the interference that ripe health check-up is surveyed, by stem ring primer in stem and the adjustment of the gradient of ring portion, the impact on the Reverse Transcription Efficiency of reverse transcription stem ring primer respectively of the structure of the structure finding out ring portion base that can be the clearest and the most definite and stem's base, is therefore easier to design the most efficient reverse transcriptase primer.
Description
Technical field
The invention belongs to detect miRNA field, particularly to the design side of the stem ring primer of a kind of reverse transcription miR-505
Method.
Background technology
At present the technology of detection miRNA mainly have Northern blot hybridization (Reinhart BJ, Weinstein EG,
Rhoades MW, et al.MicroRNAs in plants [J] .Genes Dev, 2002,16 (13): 1616^1626), micro-battle array
Row (Reinhart BJ, Weinstein EG, Rhoades MW, et al.MicroRNAs in plants [J] .Genes
Dev, 2002,16 (13): 1616^1626) and RT-PCR technology, but the most frequently used and the sensitiveest miRNA detection technique is qRT-
PCR.The fluorescence signal concentration of qRT-PCR end product depends not only on the initial expression height of miRNA, and it is also by inverse
Transcribe and the impact of PCR amplification efficiency.2005, reverse transcription quantitative PCR technique was incorporated into the detection of miRNA and works as by Chen etc.
In, establish the reverse transcription quantitative PCR technique (stem-loop RT-PCR) of entitled stem ring. he first devise one general
The reverse transcription primer of loop-stem structure, the 3 ' ends in this sequence add 5～8 bases subsequently, with 3 ' end reverse mutuals of purpose miRNA
Mend.In the presence of target miRNA, this primer is hybrid with it and carries out reverse transcription generation cDNA, adds positive anti-primer and Taqman
Probe, carries out quantitative PCR, and this loop-stem structure substantially increases efficiency and specificity (Chen C, the Ridzon D of reverse transcription
A,Broomer A J,et al.Real-time quantification of microRNAs by stem-loop RT-PCR
[J].Nucleic acids research,2005,33(20):el79-el79；Pieter Mestdagh,Tom Feys,
Nathalie Bernard,et al.High-throughput stem-loop RT-qPCR miRNA expression
profiling using minute amounts of input RNA[J].Nucleic Acids Research,2008,36
(21):el43)。
MiR-505 is positioned at mouse X-chromosome X:57647578-57647667, outside the first and second of ATP11C gene
In First Intron between aobvious son.2010, Verduci L etc. found that miR-505 can be by acting on its target ASF/
SF2 (alternative splicing factor) play the regulation and control propagation of mouse embryo fibroblasts and aging/apoptosis effect (Verduci, L,
Simili M,Rizzo M,Mercatanti A,Evangelista M et al.(2010)MicroRNA(miRNA)-
mediated Interaction between Leukemia/Lymphoma-related Factor(LRF)and
Alternative splicing factor/splicing factor2(ASF/SF2)affects mouse embryonic
Fibroblast senescence and apoptosis.J Biol Chem, 285:39551-39563), Karni R etc. sends out
Now many cells transfect ASF/SF2 can activate mTOR part signal path (Karni R, Hippo Y, Lowe SW,
Krainer AR(2008)The splicing-factor oncoprotein SF2/ASF activates mTORC1.Proc
Natl Acad Sci USA 105 (40): 15323-15327), but the concrete mode that ASF participates in regulation and control mTOR is not clear.
Yamamoto Y etc., when studying tumour MDR, find that miR-505 is new tumor suppression miRNA, present with it
The protein A kt3 of negative correlation, with the AKT on mTOR path belong to same gene family (Yamamoto Y, Yoshioka Y,
Minoura K,Takahashi R,Takeshita F et al.,(2011)An integrative genomic
analysis revealed the relevance of microRNA and gene expression for drug-
resistance in human breast cancer cells.Mol Cancer,10:13539551-39563).Therefore
MiR-505 very likely plays its biological function by regulation and control mTOR signal path, and mTOR path is current molecular biosciences
Learning the focus of research, it can play regulating cell metabolism in integrator cell and intercellular signal, growth, breeds and survival etc.
The effect of the central regulator person of process, the change of mTOR path (Mathieu Laplante and relevant to multiple disease
David M.Sabatini, mTOR signaling at a glance (2009) J Cell Sci 122,3589-3594.).
Summary of the invention
The invention provides the method for designing of the stem ring primer of a kind of reverse transcription miR-505, by using with 2005,
The general stem ring primer of the design such as Chen, as basic framework, is specific transcript with miRNA-505-3P gene, respectively
The several gradients corresponding with the design of the base number rule of ring portion by the quantity of stem's base pair of this reverse transcription stem ring primer,
Through detecting the reverse transcription of miRNA-505-3P and real time-PCR, finishing screen selects one for miRNA-505-3P
The reverse transcription stem ring primer that during gene specific reverse transcription, efficiency is optimal.
A kind of method for designing of the stem ring primer of the reverse transcription miR-505 of the present invention, including:
(1) 3 gradients of design are specific reverse transcriptase miRNA-505-during 14 pairs of base pair complementarity in fixing stem
The stem ring primer of 3P gene；
(2) 4 gradients of design are specific reverse transcriptase miRNA-505-during 21 pairs of base pair complementarity in fixing ring portion
The stem ring primer of 3P gene；
(3) amplimer of real time-PCR is designed；Wherein, forward primer 5 '-GCGAGCACCGTCAACACTTG-
3 ', downstream primer 5 '-TGCAGGGTCTGACTTATT-3 '；
(4) using the stem ring primer in (1) and (2) is CDNA by mir-505-3P gene reverse transcription；
(5) stem's structure of fixing reverse transcription stem ring primer is 14 base pairs, the ring portion structure institute reverse transcription of 3 gradients
The CDNA gone out carries out the detection of real time-PCR；PCR primer carries out electrophoresis detection；
(6) the ring portion structure of fixing reverse transcription stem ring primer is 21 base pairs, structure institute of the stem reverse transcription of 4 gradients
The CDNA gone out carries out the detection of real time-PCR；PCR primer carries out electrophoresis detection；
(7) comparison (5), (6) result, carry out the secondary detection of real time-PCR, determines the stem ring that Reversal effect is optimal
Primer.
Stem ring primer in described step (1) is respectively as follows:
5’-GTCGTATCCAGTGCAGGGTCTGACTTATAGCACTGGATACGACAGAAAACCAG-3’
5’-GTCGTATCCAGTGCAGGGTCTGACTTATTCAGGCACTGGATACGACAGAAAACCAG-3’
5’-GTCGTATCCAGTGCAGGGTCTGACTTATTCAGTACGCACTGGATACGACAGAAAACCAG-3’。
Stem ring primer in described step (2) is respectively as follows:
5’-AGTGCAGGGTCTGACTTATTCAGTACGCACTAGAAAACCAG-3’
5’-TCCAGTGCAGGGTCTGACTTATTCAGTACGCACTGGAAGAAAACCAG-3’
5’-GTATCCAGTGCAGGGTCTGACTTATTCAGTACGCACTGGATACAGAAAACCAG-3’
5’-GTCGTATCCAGTGCAGGGTCTGACTTATTCAGTACGCACTGGATACGACAGAAAACCAG-3’。
Carrying out stem ring Priming in described step (4) before reverse transcription, concrete coded program is 95 DEG C of 30s, 80 DEG C of 30s,
70℃30s,60℃30s,50℃30s,40℃30s,30℃30s,20℃30s。
Reverse transcription in described step (4) method particularly includes: stem ring primer specificity is combined in miRNA-505-3P
In sequence, it is CDNA by the specific reverse transcriptase of mir-505-3P subsequently, finally by enzyme-deactivating, cryopreservation.
The coded program of the combination of described stem ring primer specificity is 16 DEG C of 15min, the specificity of described mir-505-3P
The coded program of reverse transcription is for for 35 DEG C of 60min, 80 DEG C of 15min.
PCR reaction condition in described step (5) and (6) is: 95 DEG C of denaturations 2min, 95 DEG C of degeneration 15s, 60 DEG C of annealing
30s, 70 DEG C extend 30s, start coreaction 40 circulation from second step.
Electrophoresis detection in described step (5) and (6) uses the agarose gel of 2%.
It is contemplated that for the gene constructed the most efficient but also specific reverse transcription stem of miRNA-505 of mammal
Ring primer.For miRNA-505 gene, carry out groping of efficient reverse transcription primers by changing general stem ring primer, will be logical
First keep the invariable number of stem's base pair with stem ring primer, then gradient changes the base number of ring portion, can through subsequent detection
Best to judge the Reverse Transcription Efficiency which combines.Then the ring portion structure with preferably that group is constant, then gradient changes stem's alkali
The number of base pair, i.e. may determine that structure and the group of ring portion structure of the optimal stem base of stem ring primer through subsequent detection
Close.
The technical process of the present invention, with 2005, the general stem ring primer of the design such as Chen as basic framework, with
MiRNA-505-3P gene is specific transcript, and the pairing number first keeping stem's base of stem ring primer is constant more right
The base number of its ring portion 3 gradients of design, the most also will be at 3 ' ends plus 10 bases, with the 3 ' of purpose miRNA-505-3P
End reverse complemental, to guarantee specificity during reverse transcription.By these 3 reverse transcriptase primers simultaneously by total for mice mRNA
MiRNA-505-3P reverse transcription is CDNA；The primer of real time-PCR to be designed, the around-France forward primer of stem is relatively
Special, by 5 ' end bases (template is outer) and 3 ' distinguished sequences (in templates)) partly form, this comes from the around-France certain moduli of stem
Plate chain elongation mode.3 ' ends of forward primer have 5 ' end homologies of 12 bases and ripe body miRNA-505-3P, and primer
5 ' ends the most artificially extend 8 bases, to increase the length of purpose fragment；18 bases of downstream primer Fixed Design are at stem ring primer
Ring portion structure at, with the base homology of ring portion structure.Pass through the reverse transcription to ripe body miRNA-505-3P and real
The detection of time-PCR, with filter out Reverse Transcription Efficiency the highest time obtained stem ring portion base structure composition stem ring primer.
Keeping this stem ring primer ring portion structure constant again, stem separately designs the base pairing number of 4 gradients, through to ripe body
The reverse transcription of miRNA-505-3P and the detection of real time-PCR, may finally obtain one for miRNA-505-3P
During gene specific reverse transcription, optimal stem and the reverse transcription stem ring primer of ring portion base structure combination when efficiency is the highest.
The stem ring primer for miRNA-505-3P gene specific reverse transcription that will design, to Mouse Inferior Collicular cerebral tissue
The RNA extracted carries out reverse transcription, and respectively CDNA is diluted two gradients, carries out real time-PCR detection, to confirm it
Amplification efficiency is stable and efficient, to prove the reliability of the reverse transcription stem ring primer high efficiency of design.
Beneficial effect
(1) present invention reverse transcription to miRNA-505-3P maturation body, successfully avoids the reverse transcription to its precursor, because of
There is also hairpin structure for precursor in its natural state, therefore cannot be combined with reverse transcription primer, therefore avoid ripe body
The interference of detection；
(2) by stem ring primer in stem and the adjustment of the gradient of ring portion, can be the clearest and the most definite find out ring portion base
Structure and the structure impact on the Reverse Transcription Efficiency of reverse transcription stem ring primer respectively of stem's base, be therefore easier to design
Rationally efficient reverse transcriptase primer, for providing important reference information later when designing reverse transcriptase primer for other miRNA.
Accompanying drawing explanation
Fig. 1 is the general reverse transcription stem ring primer as skeleton；
Fig. 2 be 3 at the specific reverse transcriptase miRNA-505-3P gene that fixing stem is during 14 pairs of base pair complementarity
Stem ring primer；
Fig. 3 be 4 at the stem ring that fixing ring portion is specific reverse transcriptase miRNA-505-3P gene during 21 base numbers
Primer；
Fig. 4 is stem ring primer reverse transcription and the schematic diagram of amplification；
Fig. 5 be stem's structure of fixing reverse transcription stem ring primer be 14 base pairs, the ring portion structure of 3 gradients is reversed
The melt curve analysis of the CDNA recorded out；
Fig. 6 be stem's structure of fixing reverse transcription stem ring primer be 14 base pairs, the ring portion structure of 3 gradients is reversed
The electrophoretogram of the pcr amplification product of the CDNA recorded out；
Fig. 7 be the ring portion structure of fixing reverse transcription stem ring primer be 21 base pairs, stem's structure of 4 gradients is reversed
The melt curve analysis of the CDNA recorded out；
Fig. 8 be the ring portion structure of fixing reverse transcription stem ring primer be 21 base pairs, stem's structure of 4 gradients is reversed
The electrophoretogram of the pcr amplification product of the CDNA recorded out.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.In addition, it is to be understood that after having read the content that the present invention lectures, people in the art
The present invention can be made various changes or modifications by member, and these equivalent form of values fall within the application appended claims equally and limited
Scope.
Embodiment 1
(1) a stem ring primer skeleton is selected.(Fig. 1)
(2) for mir-505-3P gene, stem's structure of fixing reverse transcription stem ring primer is 14 base pairs, designs 3
The ring portion structure of individual gradient: (Fig. 2)
5’-GTCGTATCCAGTGCAGGGTCTGACTTATAGCACTGGATACGACAGAAAACCAG-3’
5’-GTCGTATCCAGTGCAGGGTCTGACTTATTCAGGCACTGGATACGACAGAAAACCAG-3’
5’-GTCGTATCCAGTGCAGGGTCTGACTTATTCAGTACGCACTGGATACGACAGAAAACCAG-3’。
(3) for mir-505-3P gene, the ring portion structure of fixing reverse transcription stem ring primer is 21 bases, designs 4
Stem's structure of gradient: (Fig. 3)
5’-AGTGCAGGGTCTGACTTATTCAGTACGCACTAGAAAACCAG-3’
5’-TCCAGTGCAGGGTCTGACTTATTCAGTACGCACTGGAAGAAAACCAG-3’
5’-GTATCCAGTGCAGGGTCTGACTTATTCAGTACGCACTGGATACAGAAAACCAG-3’
5’-GTCGTATCCAGTGCAGGGTCTGACTTATTCAGTACGCACTGGATACGACAGAAAACCAG-3’。
(4) amplimer of real time-PCR is designed
The forward primer of PCR is 20bp:5 '-GCGAGCACCGTCAACACTTG-3 '
The downstream primer of PCR is 18bp:5 '-TGCAGGGTCTGACTTATT-3 '.
The different PCR primer size corresponding to stem ring primer of design:
(5) extraction of mammal total serum IgE
First clip Mouse Inferior Collicular cerebral tissue puts into liquid nitrogen, is then transferred in trizol lysate, carries out under refiner
Tissue grinder, is separated to upper phase with chloroform by total serum IgE, transfers to upper phase be precipitated by total serum IgE in isopropanol reagent
Out, then carry out washing 2 times with the ethanol of 75%, dissolve with DEPC water after total serum IgE air-dries, shake, centrifugal after carry out
Concentration measures.The most rearmounted-80 DEG C of Excised Embryos.
(6) reverse transcription of mir-505-3P
First have to total serum IgE is carried out DNA enzymatic process, the DNA wherein remained with removing；Then stem ring Priming is carried out,
Concrete coded program is: 95 DEG C of 30s, 80 DEG C of 30s, 70 DEG C of 30s, 60 DEG C of 30s, 50 DEG C of 30s, 40 DEG C of 30s, 30 DEG C of 30s, 20 DEG C
30s, to allow stem ring primer correctly match.
Being undertaken in two steps reverse transcription again, first step coded program is 16 DEG C of 15min, in order to the combination of stem ring primer specificity
In mir-505-3P sequence；Second step coded program is 35 DEG C of 60min, 80 DEG C of 15min, so that stem ring primer is at reverse transcriptase
Effect under, be CDNA by the specific reverse transcriptase of mir-505-3P, finally by enzyme-deactivating.After lower machine, CDNA is put-20 DEG C of ice
Case preserves.
(7) real time-PCR:
For mir-505-3P gene, stem's structure of fixing reverse transcription stem ring primer is 14 base pairs, 3 gradients
The CDNA that ring portion structure institute reverse transcription goes out carries out the detection of real time-PCR.
CDNA dilution 5 times is carried out real-time fluorescence quantitative PCR reaction, 95 DEG C of denaturations 2min, 95 DEG C of degeneration as template
15s, 60 DEG C of annealing 30s, 70 DEG C extend 30s, start coreaction 40 circulation from second step, analyze melt curve analysis and CT value.CT
Value see table:
(sample 14-15: had 14 pairs of base compositions by stem, ring portion has the stem ring primer of 15 base compositions to be reversed
The CDNA of record.
Sample 14-18: had 14 pairs of base compositions by stem, ring portion has the stem ring primer institute reverse transcription of 18 base compositions
CDNA.Sample 14-21: had 14 pairs of base compositions by stem, ring portion has the stem ring primer institute reverse transcription of 21 base compositions
CDNA.)
From the point of view of melt curve analysis (Fig. 5), melt curve analysis is all unimodal, and Tm value is all about 78.95, illustrates that PCR draws
The specificity of thing is good.
Ensure that PCR primer amplification efficiency high and stable on the premise of, comprehensive analyze CT value it can be seen that at fixing stem
During portion's structure, changing the base number of ring portion, the CT value of several gradient experiment, all about 28.5, does not changes significantly, says
The efficiency of reverse transcription is affected little by the base number of bright ring portion.
Agarose gel with 2% carries out running electrophoresis and carries out identifying PCR primer (Fig. 6), wherein:
Swimming lane 1:Marker
Swimming lane 2:14-15: had 14 pairs of base compositions by stem, ring portion has the stem ring primer of 15 base compositions to be reversed
The CDNA of record, pcr amplification product size 62bp.
Swimming lane 3:14-18: had 14 pairs of base compositions by stem, ring portion has the stem ring primer of 18 base compositions to be reversed
The CDNA of record, pcr amplification product size 65bp.
Swimming lane 4:14-21: had 14 pairs of base compositions by stem, ring portion has the stem ring primer of 21 base compositions to be reversed
The CDNA of record, pcr amplification product size 68bp.
By electrophoresis result it can be seen that purpose fragment is between 50-100bp, illustrate that the product of specific amplification is errorless.
(8) real time-PCR:
For mir-505-3P gene, the ring portion structure of fixing reverse transcription stem ring primer is 21 bases, the stem of 4 gradients
The CDNA that structure institute of portion reverse transcription goes out carries out the detection of real time-PCR.
CDNA dilution 5 times is carried out real-time fluorescence quantitative PCR reaction, 95 DEG C of denaturations 2min, 95 DEG C of degeneration as template
15s, 60 DEG C of annealing 30s, 70 DEG C extend 30s, coreaction 40 circulation, analyze melt curve analysis and CT value.
| Sample name | C т value | CT meansigma methods | Tm1 |
| 5-21 | 25.69250298 | 26.33472125 | 77.19274902 |
| 5-21 | 25.7788372 | 77.19274902 | |
| 8-21 | 26.70027924 | 26.74422741 | 78.31764984 |
| 8-21 | 26.78817558 | 78.49358368 | |
| 11-21 | 27.40894127 | 27.38426876 | 78.49358368 |
| 11-21 | 27.35959625 | 78.49358368 | |
| 14-21 | 27.92188454 | 27.85992432 | 78.66951752 |
| 14-21 | 27.7979641 | 78.66951752 |
(sample 5-21: had 21 base compositions by ring portion, the stem ring primer that stem is made up of 5 base pairs is reversed
The CDNA of record.
Sample 8-21: there are 21 base compositions by ring portion, stem is made up of 8 base pairs stem ring primer institute reverse transcription
CDNA.Sample 11-21: had 21 base compositions by ring portion, the stem ring primer that stem is made up of 11 base pairs is reversed
The CDNA of record.
Sample 14-21: had 21 base compositions by ring portion, the stem ring primer that stem is made up of 14 base pairs is reversed
The CDNA of record.)
Coming (Fig. 7) from melt curve analysis, melt curve analysis is all unimodal, and Tm value is all about 78.67, illustrates that PCR draws
The specificity of thing is good.
Ensure that PCR primer amplification efficiency high and stable on the premise of, comprehensive analyze CT value it can be seen that in retainer ring
When portion's structure is 21bp, the logarithm of the base pair of stem is respectively with 5bp, and when 8bp, 11bp, 14bp are incremented by, its CT value is gradually to increase
Big, illustrating that stem's base pairs is the most, then the efficiency of reverse transcription is successively decreased.So can determine that when stem's structure is 5
Base pair, when ring portion is 21 bases, the efficiency of reverse transcription is the highest.
Agarose gel with 2% carries out running electrophoresis and carries out identifying PCR primer (Fig. 8), wherein:
Swimming lane 1:5-21: had 21 base compositions by ring portion, the stem ring primer that stem is made up of 5 base pairs is reversed
The CDNA of record, pcr amplification product size 59bp.
Swimming lane 2:8-21: had 21 base compositions by ring portion, the stem ring primer that stem is made up of 8 base pairs is reversed
The CDNA of record, pcr amplification product size 62bp.
Swimming lane 3:11-21: had 21 base compositions by ring portion, the stem ring primer institute that stem is made up of 11 base pairs is inverse
The CDNA transcribed, pcr amplification product size 65bp.
Swimming lane 4:14-21: had 21 base compositions by ring portion, the stem ring primer institute that stem is made up of 14 base pairs is inverse
The CDNA transcribed, pcr amplification product size 68bp.
By electrophoresis result it can be seen that purpose fragment is between 50-100bp, illustrate that the product of specific amplification is errorless.
(9) being 5,8,11,14 pairs of base pairs for stem's structure, ring portion is the stem ring primer institute reverse transcription of 21 bases
CDNA carries out 4 times of gradient dilutions, again to determine stability and the high efficiency of its amplification efficiency.
| Sample name | CT | CT meansigma methods |
| 5s-1 | 25.6925 | 25.66129 |
| 5s-1 | 25.77884 | |
| 5s-1 | 25.51254 | |
| 5s-4 | 28.13411 | 28.07503 |
| 5s-4 | 28.07049 | |
| 5s-4 | 28.02051 | |
| 8s-1 | 27.53282 | 27.58481 |
| 8s-1 | 27.63679 | |
| 8s-4 | 29.85021 | 29.90929 |
| 8s-4 | 29.96837 | |
| 11s-1 | 27.61797 | 27.70411 |
| 11s-1 | 27.69995 | |
| 11s-1 | 27.79441 | |
| 11s-4 | 30.08827 | 30.08705 |
| 11s-4 | 30.17059 | |
| 11s-4 | 30.00229 | |
| 14s-1 | 28.59109 | 28.68423 |
| 14s-1 | 29.00873 | |
| 14s-1 | 28.45287 | |
| 14s-4 | 31.01489 | 30.9885 |
| 14s-4 | 31.03336 | |
| 14s-4 | 30.91726 |
(sample 5s-1: stem's structure is 5 pairs of bases, and ring portion structure is the stem ring primer institute reverse transcription of 21 bases
CDNA dilutes 5 times.
Sample 5s-4: stem's structure is 5 pairs of bases, and ring portion structure is the CDNA of the stem ring primer institute reverse transcription of 21 bases
Dilute 20 times.
Sample 8s-1: stem's structure is 8 pairs of bases, and ring portion structure is the CDNA of the stem ring primer institute reverse transcription of 21 bases
Dilute 5 times.
Sample 8s-4: stem's structure is 8 pairs of bases, and ring portion structure is the CDNA of the stem ring primer institute reverse transcription of 21 bases
Dilute 20 times.
Sample 11s-1: stem's structure is 11 pairs of bases, and ring portion structure is the stem ring primer institute reverse transcription of 21 bases
CDNA dilutes 5 times.
Sample 11s-4: stem's structure is 11 pairs of bases, and ring portion structure is the stem ring primer institute reverse transcription of 21 bases
CDNA dilutes 20 times.
Sample 14s-1: stem's structure is 14 pairs of bases, and ring portion structure is the stem ring primer institute reverse transcription of 21 bases
CDNA dilutes 5 times.
Sample 14s-4: stem's structure is 14 pairs of bases, and ring portion structure is the stem ring primer institute reverse transcription of 21 bases
CDNA dilutes 20 times .)
By data it can be seen that
A.8s-1 the CT value CT value big about 2 than 5s-1, simultaneously the CT value of the 8s-4 CT value big about 2 than 5s-4；
B.11s-1 CT value and the CT value difference of 8s-1 are minimum, and the CT value of 11s-4 differs pole with the CT value of 8s-4 simultaneously
Little；
C.14s-1 the CT value CT value big about 1 than 11s-1, the CT value of the 14s-4 CT value than 11s-4 big 1 is left simultaneously
Right；
In sum, it can be deduced that the amplification efficiency of real time PCR is efficient stable, the CT value institute drawn is anti-
The difference that should go out, mainly reflects the height of Reverse Transcription Efficiency.Therefore can be determined that when stem's structure is 5 base pairs, ring
When portion is 21 bases, the efficiency of reverse transcription is the highest.
Claims (5)
1. a method for designing for the stem ring primer of reverse transcription miR-505, including:
(1) pass through using 5 '-GTCGTATCCAGTGCAGGGTCTGACTTATTCGCACTGGATACGAC-3 ' as basic framework,
Fixing stem is 14 pairs of complementary bases, and design ring portion structure is respectively 3 gradients of 15,18,21 not complementary bases, and 3 '
End, plus 10 base the reverse complementary sequence 5 '-AGAAAACCAG-3 ' held with miR-505-3P mature sequence 3 ', constitutes special
The stem ring primer of sexual inversion record miRNA-505-3P gene；Wherein, stem ring primer is respectively as follows:
5’-GTCGTATCCAGTGCAGGGTCTGACTTATAGCACTGGATACGACAGAAAACCAG-3’
5’-GTCGTATCCAGTGCAGGGTCTGACTTATTCAGGCACTGGATACGACAGAAAACCAG-3’
5’-GTCGTATCCAGTGCAGGGTCTGACTTATTCAGTACGCACTGGATACGACAGAAAACCAG-3’；
(2) pass through using 5 '-GTCGTATCCAGTGCAGGGTCTGACTTATTCGCACTGGATACGAC-3 ' as basic framework,
Fixing ring portion is 21 not complementary bases, and design stem structure is respectively 4 gradients of 5,8,11,14 pairs of complementary bases, and
3 ' ends, plus 10 base the reverse complementary sequence 5 '-AGAAAACCAG-3 ' held with miR-505-3P mature sequence 3 ', constitute spy
The stem ring primer of opposite sex reverse transcription miRNA-505-3P gene；Wherein, stem ring primer is respectively as follows:
5’-AGTGCAGGGTCTGACTTATTCAGTACGCACTAGAAAACCAG-3’
5’-TCCAGTGCAGGGTCTGACTTATTCAGTACGCACTGGAAGAAAACCAG-3’
5’-GTATCCAGTGCAGGGTCTGACTTATTCAGTACGCACTGGATACAGAAAACCAG-3’
5’-GTCGTATCCAGTGCAGGGTCTGACTTATTCAGTACGCACTGGATACGACAGAAAACCAG-3’；
(3) amplimer of real time-PCR is designed；Wherein, forward primer 5 '-GCGAGCACCGTCAACACTTG-3 ', under
Trip primer 5 '-TGCAGGGTCTGACTTATT-3 '；
(4) using the stem ring primer in (1) and (2) is cDNA by mir-505-3P gene reverse transcription；Carry out real time-PCR
Detection；PCR primer carries out electrophoresis detection；
(5) result corresponding with stem ring primer in (1) and (2) obtained in (4) is compared, then carry out real
The secondary detection of time-PCR, determines the stem ring primer that Reversal effect is optimal.
The method for designing of the stem ring primer of a kind of reverse transcription miR-505 the most as claimed in claim 1, it is characterised in that: described
Reverse transcription in step (4) method particularly includes: being combined in stem ring primer specificity in miRNA-505-3P sequence, subsequently will
The specific reverse transcriptase of mir-505-3P is cDNA, finally by enzyme-deactivating, and cryopreservation.
The method for designing of the stem ring primer of a kind of reverse transcription miR-505 the most as claimed in claim 2, it is characterised in that: described
The coded program of the combination of stem ring primer specificity is 16 DEG C of 15min, the upper machine of the specific reverse transcriptase of described mir-505-3P
Program is for for 35 DEG C of 60min, 80 DEG C of 15min.
The method for designing of the stem ring primer of a kind of reverse transcription miR-505 the most as claimed in claim 1, it is characterised in that: described
PCR reaction condition in step (4) is: 95 DEG C of denaturations 2min, 95 DEG C of degeneration 15s, 60 DEG C of annealing 30s, and 70 DEG C extend 30s,
Coreaction 40 circulation is started from second step.
The method for designing of the stem ring primer of a kind of reverse transcription miR-505 the most as claimed in claim 1, it is characterised in that: described
Electrophoresis detection in step (4) uses the agarose gel of 2%.
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