CN1563402A - Method for generating interference RNA in short double bond through in vitro transcription - Google Patents
Method for generating interference RNA in short double bond through in vitro transcription Download PDFInfo
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Abstract
A method for transcripting in vitro and generating short double strand interference RNA is first of all to design synthesizing promoter T7 18pb and sequence 18bp DNA oligonuclcotide strand containing target gene 20bp reversedly compensated with promoter T7 to form double-strand DNA after annealing to synthesize promoter T7 and the inversed inter-compensation sequence 20bp containing target gene and T7 target gene and its double-strand DNA strand as the temple to transcript and generate positive RNA and negative RNA to get positive and negative single strand RNA with DNA in DNase-1 degradation reaction system to be annealed and purified to get short double-strand RNA.
Description
Technical field
What the present invention relates to is the method that a kind of in-vitro transcription generates short double-chain interference RNA, and particularly a kind of method with the synthetic siRNA of t7 rna polymerase catalysis in-vitro transcription is used for biology, gene engineering technology field.
Background technology
In the transgenosis test in early days, people attempt to improve by the copy that increases certain functional gene its expression, yet some results show this genetic expression and not only do not improve reduction on the contrary that this phenomenon is known as common inhibition on plant, be known as compacting on fungi.Nineteen ninety-fives such as Su find that antisense and just RNA have blocked expression of gene in the test of expressing with sense-rna blocking-up nematode gene, they do not understand this result very much.Up to studies have shown that on nematode in 1998 such as Fire, also blocked in the test of genetic expression at just RNA, what really work is the double-stranded RNA that produces when in-vitro transcription justice RNA, and they claim that this double-stranded RNA is that RNA disturbs to the blocking effect of genetic expression.
The mechanism of action of RNAi is illustrated substantially at present, promptly when in the organism because the transcription product of picornavirus infection, transposon and external source quiding gene when producing double-stranded RNA, can bring out cell and produce immune response, activate the body rna plymerase iii, and double-stranded RNA is cut into several short double-chain interference RNAs.The same rna plymerase iii of this short double-chain interference RNA, rna helicase enzyme etc. are in conjunction with forming mixture, mixture is with the homology part combination among the mRNA under the guidance of short double-chain interference RNA, under the effect of rna plymerase iii, long-chain RNA is cut off, the result is that virus is eliminated, transposon or external source quiding gene are expressed and are blocked, simultaneously with its homologous cellular genome in genetic expression also be blocked.
The RNA interference phenomenon because can special, the expression of blocking gene efficiently, and have caused the extensive concern and the great interest in biological study field since revealed in nematode.In biologies such as fungi, Arabidopis thaliana, hydra, trypanosome, turbellarian worm, fruit bat, zebra fish, mouse, found this gene silencing phenomenon at present.In nematode,,, RNA has been used for high-throughout gene functional research so disturbing because the lead-in mode of double-stranded RNA very makes things convenient for (immersion or feeding get final product).In biologies such as fruit bat, zebra fish, mouse, also carried out the research aspect the fetal development.But be subjected to the influence of dsRNA lead-in mode and fetal development characteristics, the RNAi research in the vertebrates concentrates on the cell levels at present more.That RNAi has is simple to operate, applied widely, high specificity, can therefore be to utilize reverse genetics to understand comparatively ideal instrument of gene function in advantage such as a plurality of target genes of inactivation mutually and simultaneously simultaneously not.
Implement the RNA perturbation technique mainly contain synthetic disturb with in double-stranded RNA, double-stranded RNA transfered cell or the body, the detection of RNA interference effect and observe three partial contents, and the acquisition double-stranded RNA is the first step that carries out the RNA The Study of Interference.Especially for Mammals, long double-stranded RNA causes the non-specific restriction of several genes owing to activating body virus defense mechanism, the short dsrna ability specific inhibition expression of gene of having only 21～23 nucleotide pairs, this is to disturbing with the synthetic certain degree of difficulty that increased of double-stranded RNA.
Nearly 2 years, the foreign scholar was on the increase transcribing the research report that generates the double-chain interference RNA method in external and the body, and some have changed into commodity rapidly and have entered market, and comparing with chemical synthesis method greatly reduces experimental cost.Obtain double-chain interference RNA at present and mainly contain three kinds of ways, promptly transcribe method in chemical synthesis, in-vitro transcription method and the body.Generally be not used because the cost of chemical synthesis is too high; The basic ideas of transcribing method in the body are the carriers that can express double-stranded RNA earlier in external structure, again carrier is transferred in the cell by the spontaneous generation double-stranded RNA of transcribing of cell, in the cell strain of expression vector steady in a long-term, double-stranded RNA can be brought into play the effect of blocking gene for a long time like this.Though the method for transcribing has remarkable advantages in the body, but still be confined to minority species and clone, vector expression efficient and action rule thereof are also among research.The in-vitro transcription method has various ways, but the basic practice is to be template with one section homologous DNA sequence on the target gene mRNA, and it is taken off fiery combination with the T7 promoter primer, and under the effect of t7 rna polymerase, being transcribed into RNA then, two complementary RNA take off fire and are combined into double-stranded RNA.By prior art documents, find: Olivier Donz é and Didier Picard.RNA interference inmammalian cells using siRNAs synthesized with T7 RNA polymerase.Nucl.Acids.Res.2002,30:e46[author: Olivier Donz é and Didier Picard, title of article: with the RNA The Study of Interference of T7 RNA polymerase synthetic short double-chain interference RNA in mammalian cell; Magazine: nucleic acids research (Britain), 2002,30:e46].The document is transcribed on the stencil design at the oligonucleotide of 38 bases, and 2 bases of its 5 ' end beginning are picked at random, and 2 bases in RNA 3 ' end end that its result transcribes out also are at random.The present invention then transcribes at the oligonucleotide of 38 bases very big change on the stencil design, both 2 bases of 5 ' end beginning were chosen 2 VITAMIN B4, and 2 bases in RNA 3 ' end end that its result transcribes out are uridylics.When positive and negative two single stranded RNAs annealing of transcribing out forms double-stranded RNA, the uridylic that two ends respectively overhang.Many RNA The Study of Interference think, combinations such as the double-stranded RNA of this structure and same rna plymerase iii, rna helicase enzyme form the easier mRAN with target gene of mixture in conjunction with and cut off it, thereby more effectively strike the low target expression of gene.In addition, document directly with dissolving behind the ethanol sedimentation, does not carry out any purification to the synthetic short dsrna.The present invention then adopts the phenol-chloroform extraction process, and the impurity such as protein in most of reaction system of having dispelled out obtain highly purified short dsrna, and cell or body are to importing the specification of quality of short dsrna when having guaranteed to carry out the RNA The Study of Interference.
Summary of the invention
The objective of the invention is to be to overcome deficiency of the prior art the method that provides a kind of in-vitro transcription to generate short double-chain interference RNA, the method for particularly synthesizing short double-chain interference RNA with t7 rna polymerase catalysis in-vitro transcription.Making it will be that a cover is simple to operate, cost is low, the high short double-chain interference RNA synthetic method of efficient, on a large scale, high-throughput carries out the RNA The Study of Interference that the short double-chain interference RNA synthetic method is provided.
The present invention is achieved by the following technical solutions, the present invention at first designs synthetic T7 promotor 18bp and contains target gene 20bp and T7 promotor reverse complementary sequence 18bpDNA oligonucleotide chain, after annealing, both form double-stranded DNA, synthesize T7 promotor 18bp and contain target gene reverse complementary sequence 20bp and T7 promotor reverse complementary sequence 18bpDNA oligonucleotide chain, also form another double-stranded DNA after both annealing, the T7 RNA polymerase is under the guiding of T7 promotor, with the double-stranded DNA chain of two target genes that contain the T7 promotor and target gene reverse complementary sequence is that template is transcribed respectively and generated positive chain RNA and anti-chain RNA, product after two transcribed is with the DNA in the DNase-I DeR system, obtain positive single stranded RNA and anti-single stranded RNA, will just generate in two reaction systems, just obtain short dsrna behind negative single stranded RNA annealing and the purifying.
Method flow is as follows:
1, the selection of target gene interference position
On internet biometric database GenBank inquiry want interference base because of the mRNA sequence, 20 base sequences beginning of searching AAG are as the target gene sequence after 75 bases of atg start codon (ATG).Require G in this section sequence, C content at 30%-45%, and selected sequence is carried out homology compare (BLAST) on macro-organism databases such as GenBank, require this section sequence and other gene not to have the height homology, otherwise can not determine and be RNA jamming target sequence.
2, the design of DNA oligonucleotide chain is with synthetic
The synthetic T7 promoter DNA oligonucleotide chain (18bp) of design, article one, contain the target gene forward sequence (20bp) that AAG begins and the DNA oligonucleotide chain (38bp) of T7 promotor reverse complementary sequence (18bp), one contains the target gene reverse sequence (20bp) that AAG begins and the DNA oligonucleotide chain (38bp) of T7 promotor reverse complementary sequence (18bp).
She Ji purpose like this: at first the T7 promoter DNA oligonucleotide of 18 bases forms double-stranded DNA with DNA oligonucleotide (containing target gene homologous sequence and the T7 promotor oligonucleotide complementary sequence) annealing of 38 bases, because t7 rna polymerase only just can be transcribed template DNA when the T7 promotor is two strands, therefore the double-stranded DNA template that forms is above transcribed under the t7 rna polymerase effect and is generated RNA.
Its prior reason is, when transcribing, T7 promoter DNA oligonucleotide chain 5 ' → 3 ' last bases G, when transcribing, included in the RNA sequence by t7 rna polymerase, first base of promptly transcribing the RNA of generation is G.Because preceding 3 bases of 38 base DNA oligonucleotide chains 5 ' end are designed to AAG, the 19th, 20,21 bases of RNA of transcribing generation are CUU in addition.Therefore, the result who transcribes is that forward, the negative sense RNA single strand 5 ' end of transcribing generation all begin with G, and 3 ' end is all with CUU ending, other base sequence complementation.Respectively there is the short dsrna of 2 UU that dangle at formation base complementrity, two ends when forward, negative sense RNA single strand mixing annealing.This structure is to carry out RNA to disturb desirable.
3, the double-stranded DNA template forms and in-vitro transcription
The DNA oligonucleotide chain that synthetic T7 promoter DNA oligonucleotide chain is contained forward and reverse sequence of target gene and T7 promotor reverse complementary sequence respectively together, earlier 95 ℃ of following sex change 5 minutes, slowly cooling just obtains T7 promotor complementary double-stranded DNA then, and wherein the target gene sequence is a strand.
In the responsive transcription system, be template with two DNA chains that contain double-stranded T7 promotor and the forward and reverse sequence of strand target gene respectively, the T7 RNA polymerase is under double-stranded T7 promotor guiding, with nucleoside triphosphate mixture (rNTP) is raw material, transcribe synthetic forward single stranded RNA and reverse single stranded RNA respectively, the transcription product of this moment is DNA and RNA heterocomplex.In order to obtain single stranded RNA, experiment has obtained forward single stranded RNA and reverse single stranded RNA respectively with the DNA in the DNA I enzyme liberating reaction system.The forward single stranded RNA and the reverse single stranded RNA that obtain are mixed the back 95 ℃ of following sex change 5 minutes, hatch after 1 hour for 37 ℃ and just obtain short dsrna.
4, the separation of short double-chain interference RNA, purification
At first in the separation of short double-chain interference RNA, purification process, increased phenol/chloroform extraction steps, adopted phenol/chloroform method that synthetic product is purified, adopt anhydrous cold ethanol precipitation RNA then, with seasoning after 70% the cold ethanol washing and precipitating, with 40 μ l PBS dissolving, can obtain 20～40 μ g short double-chain interference RNAs in reaction system at every turn.Owing to contain number of chemical compositions such as protein in the reaction system, as t7 rna polymerase, DNAI enzyme, nucleoside triphosphate mixture etc., so the present invention obviously is better than prior art and can obtains 1～5 μ g short double-chain interference RNA at every turn.
The short double-chain interference RNA that the present invention obtains can strike the expression of low external source reporter gene-green fluorescent protein expression vector effectively, and the short double-chain interference RNA of transfered cell does not have nonspecific immune reaction.The present invention is simple to operate, cost is low, efficient is high, on a large scale, high-throughput carries out the RNA The Study of Interference that the short double-chain interference RNA synthetic method is provided.
Description of drawings
Fig. 1 in-vitro transcription generates the short double-chain interference RNA synoptic diagram
Among Fig. 1, when alternative scope is the full length mRNA of whole gene, generally speaking easily after opening the beginning password 75 bases seek 20 base sequences that AAG begins.
The electrophoresis detection of the synthetic short double-chain interference RNA of Fig. 2 in-vitro transcription
In Fig. 2 (a), 1,2 swimming lanes are cut product for the T7 that transcribes with the enzyme of the positive and negative double-stranded DNA of the oligonucleotide sequence annealing formation of target gene.3,4 swimming lanes are that template is transcribed the enzyme that generates RNA and cut product for the positive and negative double-stranded DNA that forms with the annealing of the oligonucleotide sequence of target gene with T7.By electrophoresis result as can be known, the DNase-I DNA chain (swimming lane 1,2) of thoroughly having degraded; The positive and negative double-stranded DNA that forms with the annealing of the oligonucleotide sequence of target gene with T7 is that the transcription product of template really is RNA (swimming lane 3,4).
In Fig. 2 (b), 1,2 swimming lanes are cut product for transcribing back DNase-I enzyme.3,4 swimming lanes are cut product for transcribing the back without the DNase-I enzyme.5 swimming lanes are for transcribing synthetic siRNA.By Fig. 2 (b) as can be seen, transcribe after the DNase-I enzyme is cut different with the mobility of cutting product without enzyme, 1, the mobility of 2 swimming lane bands is slightly larger than 3,4 swimming lane bands, may be that product length has 38bp to drop to due to the 21nt because DNase-I has degraded and transcribed template DNA.
Fig. 3 short double-chain interference RNA is to the fluoroscopic examination of chick fibroblast egfp expression influence
In Fig. 3, (a) expression represents to use the cell of cell (c) expression of green fluorescent protein expression vector and siGFP cotransfection with green fluorescent protein expression vector and siGAPDH cotransfection with green fluorescent protein expression vector cells transfected (b).Observations shows, uses green fluorescent protein normal expression in the green fluorescent protein expression vector transfection group separately; With the faint expression of green fluorescent protein in green fluorescent protein expression vector and the siGFP cotransfection group; With green fluorescent protein expression vector and siGAPDH cotransfection group green fluorescent protein normal expression.The result has proved that the method that obtains short double-chain interference RNA is effectively correct; Proved also that on the other hand there is the RNA interference mechanism in chick fibroblast, and this genetic expression is disturbed is specific.
Embodiment
Below in conjunction with summary of the invention and accompanying drawing thereof embodiment of the present invention is further specifically described:
Embodiment 1
In-vitro transcription is synthesized short double-chain interference RNA
1. transcribe Buffer and responsive transcription liquid
10 * T7 transcribes Buffer:Tris-HCl PH7.9 400mmol/L, MgCL2 60mmol/L, DTT 100mmol/L, NaCl 100mmol/L, Spermidine 20mmol/L).
Responsive transcription liquid: 10 * T7 transcribes Buffer 5 μ l, 1mmol/L rNTP Mixture, and 40U RNaseInhibitor, 100U T7 RNA polymerase, 200pmol dsDNA, 0.1U YeastPyrophosphatase adds DEPC treating water to 50 μ l.
2.DNA oligonucleotide chain is synthetic and double-stranded DNA obtains
Synthetic T7 oligonucleotide of design and target oligonucleotide (see figure 1).In 50 μ l TE Buffer (10mMTris-HCI pH8.0,1mmol/L EDTA), every adds 1nmol, hatches 2 minutes for 95 ℃, turns off heat channel, and slowly cooling forms double-stranded DNA
3. responsive transcription and short double-chain interference RNA generate
(each 50 μ l) hatch 2h in 37 ℃ with responsive transcription liquid.Add 5U RNase-Free DNase-I, hatch 15min for 37 ℃.Two pipes are mixed (100 μ l), hatch 5min for 95 ℃.Hatch 1hr for 37 ℃.
4. short double-chain interference RNA separates purification
Phenol/chloroform/the primary isoamyl alcohol (25: 24: 1) that adds 100 μ l (equivalent), the centrifugal 10min of 12000g behind the thorough mixing gets the upper strata water in another centrifuge tube.Chloroform/the primary isoamyl alcohol (24: 1) that adds 100 μ l (equivalent), the centrifugal 10min of 12000g behind the thorough mixing gets the upper strata water in another centrifuge tube.The 3M NaOCA (pH5.2) that adds 10 μ l.The cold dehydrated alcohol that adds 2.5 times of amounts is placed 30min for-20 ℃, and the centrifugal 10min of 12000g reclaims precipitation, and seasoning after 70% the cold ethanol washing and precipitating is dissolved with 40 μ l PBS.
5. in-vitro transcription is synthesized the electrophoresis detection of short double-chain interference RNA
Because the siRNA fragment is less, electrophoretic mobility difference is little between single stranded RNA and siRNA, therefore be not easy to distinguish with agarose gel electrophoresis, and bother relatively with the polyacrylamide gel electrophoresis operation, in addition, the as easy as rolling off a log degraded of single stranded RNA makes to rely on the synthetic situation of electrophoresis detection siRNA to be not easy to accomplish.The present invention with agarose gel electrophoresis detect transcribe before and transcribe after the product of DNA I enzymic digestion (Fig. 2 a), as a result 1,2 swimming lanes are not seen any band, 3,4 swimming lanes detect visible clear band at once behind dnase digestion, but it is extremely unstable that this is transcribed, enzyme is cut after product, at room temperature places more than the 30min, just detects less than electrophoretic band.Detected result proof responsive transcription is successfully transcribed out single stranded RNA.The after product enzyme is cut and annealing result shows (Fig. 2 b) to transcribing, 1,2 swimming lanes are transcribed back DNase-I enzyme and are cut product and 3,4 swimming lanes and transcribe afterwards that to cut the mobility of electrophoretic band of product without the DNase-I enzyme different, both the mobility of 1,2 swimming lane bands was slightly larger than 3,4 swimming lane bands, its reason may be that product length has 38bp to drop to due to the 21nt because DNase-I has degraded and transcribed template DNA.In addition, 5 swimming lanes are two single stranded RNAs annealing after products, and its electrophoretic band mobility is transcribed back DNase-I enzyme with 1,2 swimming lanes, and to cut product-single stranded RNA different, and possible reason is due to double-stranded RNA and the single stranded RNA difference structurally.The present invention of electrophoresis detection presentation of results successfully transcribes out RNA, and the single stranded RNA that obtains after the DNase-I enzyme is cut has generated short double-chain interference RNA really after annealing.
Embodiment 2
The synthetic short double-chain interference RNA of in-vitro transcription disturbs chick fibroblast external source green fluorescent protein to express
1. in-vitro transcription is synthesized short double-chain interference RNA
By the synthetic short double-chain interference RNA method requirement synthetic DNA oligonucleotide sequence of in-vitro transcription, T7promoter:5 '-taatacgactcactatag-3 '; SiGFP:Sense 5 '-aagaacggcatcaaggtgaactatagtgagtcgtatta-3 ', Antisense 5 '-aagttcaccttgatgccgttctatagtgagtcgtatta-3 ' (Accession No:U55763); SiGAPDH:Sense 5 '-aagcgtgttatcatctcagcctatagtgagtcgtatta-3 ', Antisense 5 '-aaggctgagatgataacacgctatagtgagtcgtatta-3 ' (Accession No:K01458).Concrete operations and reaction system composition are seen embodiment 1.
2. chick fibroblast is cultivated
Get 8-10 days fertile egg of hatching, clean the egg surface, carefully take out the chicken embryo and be put in the sterile petri dish, remove head and internal organ with the tincture of iodine or 75% alcohol.Idiosome cleans 3 times with D-Hanks liquid or PBS liquid, is cut into the fritter of 1 ~ 2mm, transfers in the moderate triangular flask of volume.Add an amount of Digestive system (0.25% trypsinase, 0.3mg/ml collagenase, 100IU/ml penicillin, 100ug/ml Streptomycin sulphate), general per 10 chicken embryos add the 20ml Digestive system, the sealing bottleneck.Room temperature stirred at a slow speed 1 hour on magnetic stirring apparatus.Add a small amount of foetal calf serum passivation trypsinase.By 100 order stainless steel meshs, prepare cell suspension then.Cell suspension is transferred in the centrifuge tube.Centrifugal 10 minutes of 800r/min.Abandon supernatant liquor.Cell precipitation is cleaned 2 times with D-Hanks liquid or PBS liquid.Add nutrient solution, make cell suspension with suction pipe piping and druming.The numeration cell.With 1 * 10
6The density inoculating cell of individual/ml.At 37 ℃, 5%CO
2Under the condition in CO
2Cultivate in the incubator.
3. green fluorescent protein expression vector and short double-chain interference RNA transfectional cell
Cell transfecting reagent is Lipofectamine TM2000 (Invitrogen).Explanation is carried out transfection in following ratio according to transfection reagent: dilution 4 μ g nucleic acid in 250 μ l cell culture fluids, and standby after mixing gently.Dilution 10 μ l cell transfecting reagent in 250 μ l cell culture fluids are at room temperature hatched 5min after mixing gently.At room temperature hatch 20min after last two kinds of diluents are mixed gently and form complex body (500 μ l).Complex body is added in the 6 hole culture dish that cell compiles more than 50%, shake mixing gently.37 ℃ of 5%CO
2Under the condition in CO
2Can observe the transfection effect after cultivating 12h in the incubator.The green fluorescent protein expression vector is pEGFP-C1Vector (Clontech).
4. behind the cell transfecting transfection nucleic acid 24h, be to observe, count, take pictures under the 488nm at fluorescent microscope (Nikon E600) white light and excitation wavelength respectively.
5. short double-chain interference RNA is to the influence of green fluorescent protein expression
Observe under fluorescent microscope behind the transfectional cell 24h, transfection and interference effect are seen accompanying drawing 3.In accompanying drawing 3, (a) expression represents to use the cell of cell (c) expression of green fluorescent protein expression vector and siGFP cotransfection with green fluorescent protein expression vector and siGAPDH cotransfection with green fluorescent protein expression vector cells transfected (b).
Observations shows, uses green fluorescent protein normal expression in the green fluorescent protein expression vector transfection group separately; With the faint expression of green fluorescent protein in green fluorescent protein expression vector and the siGFP cotransfection group; With green fluorescent protein expression vector and siGAPDH cotransfection group green fluorescent protein normal expression.The result has proved that method that the present invention obtains short double-chain interference RNA is correct, effectively; Proved also that on the other hand there is the RNA interference mechanism in chick fibroblast, and this genetic expression is disturbed is specific.
Claims (7)
1. an in-vitro transcription generates the method for short double-chain interference RNA, it is characterized in that, at first design is synthesized T7 promotor 18bp and is contained target gene 20bp and T7 promotor reverse complementary sequence 18bpDNA oligonucleotide chain, after annealing, both form double-stranded DNA, synthesize T7 promotor 18bp and contain target gene reverse complementary sequence 20bp and T7 promotor reverse complementary sequence 18bpDNA oligonucleotide chain, also form another double-stranded DNA after both annealing, t7 rna polymerase is under the guiding of T7 promotor, with the double-stranded DNA chain of two target genes that contain the T7 promotor and target gene reverse complementary sequence is that template is transcribed respectively and generated positive chain RNA and anti-chain RNA, product after two transcribed is with the DNA in the DNase-I DeR system, obtain positive single stranded RNA and anti-single stranded RNA, will just generate in two reaction systems, just obtain short dsrna behind negative single stranded RNA annealing and the purifying.
2. in-vitro transcription according to claim 1 generates the method for short double-chain interference RNA, it is characterized in that, below further specifies as follows to idiographic flow of the present invention:
(1) selection of target gene interference position
On internet biometric database GenBank inquiry want interference base because of the mRNA sequence, 20 base sequences beginning of searching AAG are as the target gene sequence after 75 bases of atg start codon (ATG);
(2) design of DNA oligonucleotide chain is synthetic
The synthetic T7 promoter DNA oligonucleotide chain (18bp) of design, article one, contain the target gene forward sequence (20bp) that AAG begins and the DNA oligonucleotide chain (38bp) of T7 promotor reverse complementary sequence (18bp), one contains the target gene reverse sequence (20bp) that AAG begins and the DNA oligonucleotide chain (38bp) of T7 promotor reverse complementary sequence (18bp);
(3) the double-stranded DNA template forms and in-vitro transcription
It is respectively with the DNA oligonucleotide chain that contains forward and reverse sequence of target gene and T7 promotor reverse complementary sequence with synthetic T7 promoter DNA oligonucleotide chain that the double-stranded DNA template forms, earlier 95 ℃ of following sex change 5 minutes, slowly cooling just obtains T7 promotor complementary double-stranded DNA then, wherein the target gene sequence is a strand
In in-vitro transcription, be template with two DNA chains that contain double-stranded T7 promotor and the forward and reverse sequence of strand target gene respectively, t7 rna polymerase is under double-stranded T7 promotor guiding, with nucleoside triphosphate mixture (rNTP) is raw material, transcribe synthetic forward single stranded RNA and reverse single stranded RNA respectively, the transcription product of this moment is DNA and RNA heterocomplex;
(4) separation of short double-chain interference RNA, purification
At first in the separation of short double-chain interference RNA, purification process, increased phenol/chloroform extraction steps, adopted phenol/chloroform method that synthetic product is purified, adopt anhydrous cold ethanol precipitation RNA then, with seasoning after 70% the cold ethanol washing and precipitating, with 40 μ l PBS dissolving, can obtain 20～40 μ g short double-chain interference RNAs in reaction system at every turn.
3. in-vitro transcription according to claim 2 generates the method for short double-chain interference RNA, it is characterized in that, with described double-stranded DNA is that the template in-vitro transcription forms DNA and RNA hybridization chain, in order to obtain single stranded RNA, experiment has obtained forward single stranded RNA and reverse single stranded RNA respectively with the DNA in DNAI enzyme liberating DNA and the RNA hybridization chain.
4. according to the method for claim 2 or 3 described in-vitro transcription generation short double-chain interference RNAs, it is characterized in that, 95 ℃ of following sex change 5 minutes, hatch after 1 hour for 37 ℃ and just obtain short dsrna after forward single stranded RNA and the reverse single stranded RNA that obtains mixed
5. generate the method for short double-chain interference RNAs according to claim 1 or 2 described in-vitro transcription, it is characterized in that, when transcribing, T7 promoter DNA oligonucleotide chain 5 ' → 3 ' 18 bases of last bases G-Di, included in the RNA sequence by the T7 RNA polymerase, first base of promptly transcribing the RNA of generation is G, preceding 3 bases of 38 base DNA oligonucleotide chains 5 ' end are designed to AAG, transcribe the RNA the 19th of generation, 20,21 bases are CUU, the result who transcribes is the forward of transcribing generation, negative sense RNA single strand 5 ' end all begins with G, 3 ' end is all with CUU ending, other base sequence complementation.
6. in-vitro transcription according to claim 5 generates the method for short double-chain interference RNA, it is characterized in that, respectively there is the short dsrna of 2 UU that dangle at formation base complementrity, two ends when forward, negative sense RNA single strand mixing annealing.
7. in-vitro transcription according to claim 5 generates the method for short double-chain interference RNA, it is characterized in that, short double-chain interference RNA can hang down the expression of external source reporter gene-green fluorescent protein expression vector, and the short double-chain interference RNA of transfered cell does not have nonspecific immune reaction.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305512A (en) * | 2013-02-05 | 2013-09-18 | 武汉上成生物科技有限公司 | Method for synthesizing double-stranded RNA used for inhibiting growth of Chilo suppressalis and using acy1-CoA desaturase SexiVPAE genes |
| CN103993002A (en) * | 2013-02-19 | 2014-08-20 | 百奥迈科生物技术有限公司 | New production process for large-scale synthesis of long-chain RNA drugs |
| CN113604524A (en) * | 2021-08-05 | 2021-11-05 | 上海交通大学 | Method for site-specific labeling of RNA |
| CN115807048A (en) * | 2022-07-19 | 2023-03-17 | 中国海洋大学 | Method for preparing double-stranded RNA |
-
2004
- 2004-03-18 CN CNA2004100170193A patent/CN1563402A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305512A (en) * | 2013-02-05 | 2013-09-18 | 武汉上成生物科技有限公司 | Method for synthesizing double-stranded RNA used for inhibiting growth of Chilo suppressalis and using acy1-CoA desaturase SexiVPAE genes |
| CN103993002A (en) * | 2013-02-19 | 2014-08-20 | 百奥迈科生物技术有限公司 | New production process for large-scale synthesis of long-chain RNA drugs |
| CN113604524A (en) * | 2021-08-05 | 2021-11-05 | 上海交通大学 | Method for site-specific labeling of RNA |
| CN113604524B (en) * | 2021-08-05 | 2023-11-24 | 上海交通大学 | Method for site-specific labeling of RNA |
| CN115807048A (en) * | 2022-07-19 | 2023-03-17 | 中国海洋大学 | Method for preparing double-stranded RNA |
| CN115807048B (en) * | 2022-07-19 | 2024-03-22 | 中国海洋大学 | Method for preparing double-stranded RNA |
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