CN106399488A - M6A single base site identification method of gene mRNA - Google Patents
M6A single base site identification method of gene mRNA Download PDFInfo
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- CN106399488A CN106399488A CN201610799660.XA CN201610799660A CN106399488A CN 106399488 A CN106399488 A CN 106399488A CN 201610799660 A CN201610799660 A CN 201610799660A CN 106399488 A CN106399488 A CN 106399488A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Abstract
The invention discloses an m6A single base site identification method of the gene mRNA. The m6A single base site identification method of the gene mRNA comprises the following steps: (1) carrying out high-throughput sequencing on a sample, picking out the target gene according to the sequencing result, and determining the approximate position of the m6A peak of the target gene; (2) obtaining all the information possibly containing the m6A site in the regional sequence of the m6A peak of the gene by utilizing the on-line tool SRAMP; (3) constructing an over-expression vector and a mutation vector of the gene; (4) transfecting cells with the two vectors obtained in the step (3), collecting the cells, and extracting RNA; and (5) carrying out inverse transcription on the RNA containing m6A in an enriching manner obtained in the step (4) to obtain cDNA, designing fluorescent quantitative primers for all the mutation site regions, comparing the fluorescent quantitation results of the over-expression vector group and the mutation vector group, and if the result of the mutation vector group is smaller than that of the over-expression vector group at one site, and P is smaller than 0.05, judging that the site is the m6A site.
Description
Technical field
The invention belongs to biological technical field, it is related to a kind of m of gene mRNA6A single base site authentication method.
Background technology
m6A refers to occur the modification that methylates on the 6th N atom of base A to be that a kind of modal post-transcriptional level is repaiied
Decorations, constitute about 2/3rds of whole RNA modifications.In eukaryote, m6A constitutes about the whole adenosine content of cell mRNA
0.1%-0.4%.With the high speed development of secondary sequencing technologies, each biological genome storehouse is progressively set up completely, is come using sequencing
The methylating of detection gene is distributed and filters out methylation differential gene has become the preferable important side studying gene methylation
Formula.This laboratory establishes mRNA Methylation high-flux detection method it is achieved that multiple sample full transcript profile methylation
Relatively, to mRNA m6The multifarious biological study of A opens new technology.But m6A can not affect its pairing base
Ability, and do not have chemical reagent to change or this modification of specific marker, prior art cannot accurately determine m6A site.This
Invention on the basis of high-flux sequence, using site estimation and binding molecule biology techniques can realize single base
m6A site precise Identification, this is to study m further6The function of A provides technical guarantee.
Content of the invention
In order to solve the above problems, the present invention provides the m of a gene mRNA6A single base site authentication method, including with
Lower step：
1), basis《MRNA Methylation high-flux detection method》The method set up is to sample m6A high-flux sequence, according to survey
Sequence result finds target gene, and determines its m6A peak approximate location；
2), utilize online tool SRAMP (http://www.cuilab.cn/sramp/) obtain the m of this gene6A peak area
There may exist m in the sequence of domain6A site；
3), by all possible m6A site carries out point mutation, and the CDS sequence of this gene and mutant nucleotide sequence are connected respectively
Enter mammalian expression vector pSilencer4.1, build overexpression vector and the mutational vector of this gene；
4), by step 3) in two kinds of carriers transfectional cells respectively of obtaining, collect cell extraction RNA；The RNA piece that will extract
Duan Hua, using immunoprecipitation, obtain enrichment contains m6The RNA fragment of A；
5), by step 4) enrichment that obtains contain m6The RNA reverse transcription of A is cDNA, designs each mutational site region
Fluorescent quantitation primer；By the fluorescent quantitation results contrast of overexpression vector group and mutational vector group, if certain site mutation carrier
Group expression is less than overexpression vector group and P<0.05 (t-test), then judge that this site is m6A site.
M as the gene mRNA of the present invention6The improvement of A single base site authentication method, target gene is FAM134B base
Cause, its mutation (samesense mutation) be：
Site 1：GGACA→GGCCA
Site 2：TGACC→TGCCC
Site 3：GGACA→GGCCA；
Fluorescence quantification PCR primer is：
Site 1：F 5’-ACGGGACCTTCAACCTTTCA-3’
R 5’-AGTGCCTCTCTTTGCTTGGT-3’
Site 2：F 5’-CCAAGCAAAGAGAGGCACTCA-3’
R 5’-CTAACTGGTCTTTGATGGCGG-3’
Site 3：F 5’-CCGCCATCAAAGACCAGTTAG-3’
R 5’-TGGCCTCCGAGTAGATTTGA-3’.
M as the gene mRNA of the present invention6The improvement further of A single base site authentication method：
Described step 1)：According to 2016103629813 invention《MRNA Methylation high-flux detection method》Carry out high pass
Measure sequence；Target gene is picked out according to sequencing result, and determines its m6A peak approximate location.
The invention provides a kind of fast and convenient method, detect individual gene all possible methylation characteristic sequence
(RRACH) m in6The particular location that A site is located.The present invention utilizes simple and quick reliable biology techniques method first, really
Determine m6A single base site；The present invention can be for realizing m6The biological function research in A single base site provides important technology to support.
Brief description
Below in conjunction with the accompanying drawings the specific embodiment of the present invention is described in further detail.
Fig. 1：Landrace back fat (C-LB) tissue m6A sequencing gained FAM134B gene methylation distribution (m6A peak region
1250-1490bp)；
Fig. 2：FAM134B sequencing result m6M in A peak region6A site；
Fig. 3：M for FAM134B6A peak regional sequence (underlined sequences be possible the codon containing m6A)；
Fig. 4：The m of FAM134B6Sequence (underlined sequences are the codon after mutation) after A peak region mutagenesis
Fig. 5：Fluorescent quantitation result shows the m that site 3 is FAM134B gene6A site.
Specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1,
1st, according to 2016103629813 invention《MRNA Methylation high-flux detection method》Carry out high-flux sequence；Root
Selecting pig FAM134B according to sequencing result is target gene (as shown in Figure 1), and determines its m6A peak approximate location is region 1250-
At 1490bp.
2nd, search for and obtain the sequence in this region on NCBI.Enter SRAMP website (http://www.cuilab.cn/
Sramp/), select prediction, by sequence inputting in the Mature mRNA mode frame on right side, select submit, obtain
All of m at pig FAM134B gene 1250-1490bp6A predicts site.
3rd, by possible m at 36A site carries out point mutation, respectively：
Site 1：GGACA→GGCCA
Site 2：TGACC→TGCCC
Site 3：GGACA→GGCCA
By the CDS sequent synthesis of mutation at 3 and be connected on mammalian expression vector pSilencer4.1, build
FAM134B mutational vector.In addition, by the original CDS sequence of FAM134B and being connected to mammalian expression vector
On pSilencer4.1, it is built into FAM134B overexpression vector as comparison.
4th, culture people source embryonic kidney cell system (293T), when the cell of 6 10cm plates all grows to 80-90% density,
Carry out the transfection of carrier, overexpression vector and each transfection of mutational vector 3 with lipofectamine lipofectamine3000
Plate, comprises the following steps that：
1), according to transfection when density be 80-90% about requirement, day before transfection by culture medium replace with nonreactive culture
Base.
Remarks explanation：Culture 293T cell used medium is the DMEM containing 10% hyclone and 1% mycillin
Basic culture medium.The nonreactive culture medium replaced is the DMEM Basic culture medium containing 10% hyclone.Hyclone is purchased from
Hangzhou Ilex purpurea Hassk.[I.chinensis Sims biology company limited, mycillin is purchased from Gibco company with DMEM Basic culture medium.
2), each ware cell all proceeds as follows：Dilute respectively using DMEM Basic or OPTI-MEM culture medium 0.5ml
Release 10ug FAM134B overexpression vector/mutational vector and 25uL lipofectamine3000 transfection reagent, 37 DEG C of placements
5min mixes, and after room temperature preservation 15min, complexes drop-wise is added in ware.
Lipofectamine3000 transfection reagent is purchased from life biology company limited.
3), after, jiggling cell, it is put in CO26h (37 degrees Celsius of constant temperature, 5%CO is cultivated in incubator2)；Change nonreactive
Complete medium.After transfection 24h, collect cell.
5th, cell methylate mRNA extract
The cell of step 4 gained is extracted the total serum IgE of sample with Trizol method, concrete grammar is：
1. cell is placed in the 1.5ml eppendorf pipe of RNase-free；
2. 1mlTrizol is added to jiggle greatly to uniform solution；
3. after adding 200ul chloroform, jiggle greatly to uniform solution, 4C, 12000g are centrifuged 15min；
4. take supernatant in the 1.5ml eppendorf pipe of another RNase-free, add same volume isopropanol, gently
Light mixing, incubated at room 10min, 4C, 12000g are centrifuged 10min；
5. remove supernatant, with 1ml 75% washing with alcohol precipitation, 4C, 7500g are centrifuged 5min
6. 20-50ul RNase-free H2O is used to dissolve.
The total serum IgE of upper step gained is extracted mRNA with Genelute mRNA miniprep Kits (Sigma), specifically side
Method is：
1. total RNA volume is expanded to 250ul, add 250ul binding solution, mix；
2. add 15ul beads, jiggle greatly to uniform solution, after being placed in 70C incubation 3min, be placed in room temperature 10min；
3. maximum velocity centrifugation 2min, discards supernatant；
4. use the resuspended beads of 500ul wash buffer, and be transferred to spin filter；
5. it is centrifuged 1-2min, discard flow through；
6. repeat step is 4., 5.；
7. the Elution Buffer 50ul being previously heated to 70C, 70C is added to be incubated 2-5min；
8. centrifugation product is mRNA；
9. by gained mRNA using being concentrated in vacuo to 9ul.
6th, by total mRNA through ultrasonic fracture or metal ion (as Zn2+、Ca2+、Fe2+Deng) fragment into 100～200nt
MRNA fragment, under concrete grammar enters：
RNA fragmentation system
Temperature, incubation time are：70 DEG C, 15min, add EDTA terminating reaction.
Fragmentation buffer (10X) is：800 μ L RNase-free water, 100 μ L 1M Tris-HCl (pH=7.0), 100
μL 1M ZnCl2Solution.
7th, immunoprecipitation
1), the mRNA of fragmentation is taken out 50ng about as Input (comparison), remainder is defined as IP, and (immunity is sunk
Form sediment), first with immunoprecipitation, with m6A antibody is incubated 2 hours under 4 DEG C of low temperature, comprises the following steps that：
It is incubated 2h at 4 DEG C.
2), closing beads (Protein A, Life technologies, 10002D)
40 μ l beads are taken to be placed on magnetic frame, supernatant discarded, washed three times with the 1*IP buffer of 1ml, be resuspended in 1*IP
In closing buffer 1ml, it is incubated 2h in 4 DEG C.1*IP closing buffer is formulated as follows：
| 5×IP buffer | 200μl |
| RNAase inhibitor | 10μl |
| BSA | 25μl |
| RNAase free water | 765μl |
| Total | 1000μl |
After incubation 2h, washed three times with the 1*IP buffer of 1ml, resuspended with 100 μ l 1*IP buffer.
3), beads and mRNA- antibody mediated immunity precipitation：
By above-mentioned 1) and 2) sample mix that obtains, 4 DEG C of incubation 2h.
4), eluting I
By step 3) gained sample is placed on magnetic frame, abandons supernatant, washed three times with 500 μ l 1*IP buffer, be resuspended in
Eluting buffer 450ul, is placed in 4 DEG C of incubation 1h.Eluting buffer is formulated as follows：
| 5×IP buffer | 90μl |
| RNAase inhibitor | 7μl |
| M6A | 150μl |
| RNAase free water | 203μl |
| Total | 450μl |
5), eluting II
By 4) gained sample is placed on magnetic frame, collects supernatant；
Then add 50ul eluting buffer in beads, be placed in 4 DEG C of incubation 30min, be again placed on magnetic frame,
Collect supernatant again；
6), by step 5) 2 parts of supernatants merge, obtain 200ul eluent (RNA eluent).
7), ethanol precipitation：By 200ul RNA eluent+20ul NaAc (concentration be 3M)+500ul ethanol (100%)+
4ul glycogen (5mg/ml), -80 DEG C, overnight precipitation.
8), at 4 DEG C, 14000rpm is centrifuged 30min within second day, abandons supernatant, adds 1ml75% ethanol at 4 DEG C, 14000rpm
Centrifugation 30min, abandons supernatant, after air-drying, adds 9ul RNase-free water, and measures concentration.
Thus obtaining the RNA fragment containing m6A of enrichment.
8th, design of primers
The RNA reverse transcription containing m6A of the enrichment that step 7 is obtained is cDNA, and the fluorescence in design mutational site region is fixed
Amount primer：According to the mutational site regional sequence providing on NCBI, covering RRACH (R=G, the A methylating highly conserved；H
=A, C, T) sequence area design fluorescence quantification PCR primer (for multipair fluorescence quantification PCR primer), for analyzing the m of gene6A
Distribution.The primer in each site：
Site 1：F 5’-ACGGGACCTTCAACCTTTCA-3’
R 5’-AGTGCCTCTCTTTGCTTGGT-3’
Site 2：F 5’-CCAAGCAAAGAGAGGCACTCA-3’
R 5’-CTAACTGGTCTTTGATGGCGG-3’
Site 3：F 5’-CCGCCATCAAAGACCAGTTAG-3’
R 5’-TGGCCTCCGAGTAGATTTGA-3’
9th, real-time fluorescence relative quantification PCR
Overexpression vector group (FAM) is become cDNA with the RNA reverse transcription of mutant test group (mF), routinely fluorescent quantitation
PCR step operation.Using the target gene Ct difference of m6A-IP and Input as reference, interpretation of result adopts 2-△△CtMethod, its
The computing formula of middle △ △ Ct is as follows：△ △ Ct=(CtmF-CtCon)X1-(CtmF-CtCon)X2
X1 and X2 represents any two sample to be compared, calculates, by above-mentioned formula, the target gene first that X1 is with respect to X2
Base relative level.As shown in figure 5, obtaining with the FAM134B expression of overexpression vector group (FAM) for " 1 ", mutation tries result
Test the variable block diagram of the FAM134B expression of group (mF).
Result shows, the FAM134b expression mutation group in site 3 is substantially less than overexpression group, other sites no significance difference
Different, because the carrier that mutant test group (mF) transfects entrance cell will not produce containing m6The FAM134B RNA fragment of A, therefore
Mutant test group (mF) the enrichment m obtaining in step 76Do not contain FAM134B in the RNA of A, lead to this group in fluorescent quantitation result
Theoretical value can decline.
In conjunction with m6A sequencing gained FAM134B gene methylation scattergram it was demonstrated that site 3 (1356-1361) is Landrace
The methylation sites of FAM134B gene.
Comparative example 1, the primer in embodiment 1 step 8 is made into as follows：
Site 1：F 5’-ACCGCTCGATGAGTGATCCTTACC-3’
R 5’-AAACGGTAAGGATCACTCATCGAG-3’
Site 2：F 5’-CCAGGCCTATGACGATGGAG-3’
R 5’-ACCGTCGCACGATATCACTC-3’
Site 3：F 5’-AATCAAGGCTGTTGAACCCG-3’
R 5’-TCCTGTGAACACCTGCTGAT-3’
Remaining content is equal to embodiment 1.
In fluorescent quantitation result, solubility curve is abnormal, shows that primer specificity is not good.This result is unavailable.
Last in addition it is also necessary to it is noted that listed above be only the present invention several specific embodiments.Obviously, this
Bright be not limited to above example, can also have many deformation.Those of ordinary skill in the art can be from present disclosure
The all deformation directly derived or associate, are all considered as protection scope of the present invention.
Claims (3)
1. the m of gene mRNA6A single base site authentication method is it is characterised in that comprise the following steps：
1), sample is carried out high-flux sequence, target gene is picked out according to sequencing result, and determine its m6A peak approximate location；
2) m of this gene, is obtained using online tool SRAMP6There may exist m in A peak regional sequence6A site information；
3), by all possible m6A site carries out point mutation, and by the CDS sequence of this gene and base through the mutation changing
CDS sequence is connected into mammalian expression vector pSilencer4.1 respectively, builds overexpression vector and the mutational vector of this gene；
4), by step 3) two kinds of carriers transfectional cells respectively of obtaining, collect cell extraction RNA；The RNA fragmentation that will extract,
Using immunoprecipitation, obtain enrichment contains m6The RNA fragment of A；
5), by step 4) enrichment that obtains contain m6The RNA reverse transcription of A is cDNA, designs the fluorescence in each mutational site region
Quantitative primer；By the fluorescent quantitation results contrast of overexpression vector group and mutational vector group, if certain site mutation vehicle group result
Less than overexpression vector group and P<0.05, then judge that this site is m6A site.
2. the m of gene mRNA according to claim 16A single base site authentication method it is characterised in that：
Target gene is FAM134B gene, and it sports：
Site 1：GGACA→GGCCA
Site 2：TGACC→TGCCC
Site 3：GGACA→GGCCA；
Fluorescence quantification PCR primer is：
Site 1：F 5’-ACGGGACCTTCAACCTTTCA-3’
R 5’-AGTGCCTCTCTTTGCTTGGT-3’
Site 2：F 5’-CCAAGCAAAGAGAGGCACTCA-3’
R 5’-CTAACTGGTCTTTGATGGCGG-3’
Site 3：F 5’-CCGCCATCAAAGACCAGTTAG-3’
R 5’-TGGCCTCCGAGTAGATTTGA-3’.
3. the m of gene mRNA according to claim 16A single base site authentication method it is characterised in that：
Described step 1)：According to 2016103629813 invention《MRNA Methylation high-flux detection method》Carry out high pass measurement
Sequence；Target gene is picked out according to sequencing result, and determines its m6A peak approximate location.
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Cited By (4)
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| CN107385078A (en) * | 2017-08-30 | 2017-11-24 | 浙江大学 | Label of pig fat deposition description correlation PNPLA2 mRNA m6A methylate the authentication method and application of function of unit point |
| CN107419026A (en) * | 2017-08-30 | 2017-12-01 | 浙江大学 | Label of pig fat deposition description correlation UCP2 mRNA m6A methylate the authentication method and application of function of unit point |
| CN114480670A (en) * | 2022-01-26 | 2022-05-13 | 西北农林科技大学 | DNA methylase TET1 and m related to bovine skeletal muscle differentiation6Interaction identification and application of A methylase METTL3 |
| CN114592067A (en) * | 2022-01-26 | 2022-06-07 | 西北农林科技大学 | Bovine skeletal myogenesis associated MEF2C mRNA m6Identification and functional use of A methylation sites |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107385078A (en) * | 2017-08-30 | 2017-11-24 | 浙江大学 | Label of pig fat deposition description correlation PNPLA2 mRNA m6A methylate the authentication method and application of function of unit point |
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| CN107419026B (en) * | 2017-08-30 | 2020-12-22 | 浙江大学 | Identification method and functional application of UCP2mRNA m6A methylation unit site related to pig fat deposition |
| CN114480670A (en) * | 2022-01-26 | 2022-05-13 | 西北农林科技大学 | DNA methylase TET1 and m related to bovine skeletal muscle differentiation6Interaction identification and application of A methylase METTL3 |
| CN114592067A (en) * | 2022-01-26 | 2022-06-07 | 西北农林科技大学 | Bovine skeletal myogenesis associated MEF2C mRNA m6Identification and functional use of A methylation sites |
| CN114480670B (en) * | 2022-01-26 | 2022-12-06 | 西北农林科技大学 | DNA methylases TET1 and m related to bovine skeletal muscle differentiation 6 Identification and application of A methylase METTL3 interaction |
| CN114592067B (en) * | 2022-01-26 | 2023-03-28 | 西北农林科技大学 | Bovine skeletal myogenesis associated MEF2C mRNA m 6 Identification and functional use of A methylation sites |
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Application publication date: 20170215 |