CN110004142A - The method and its application of mRNA ribosomes new polypeptide chain compound are quickly completely obtained using molecular sieve centrifugal column - Google Patents
The method and its application of mRNA ribosomes new polypeptide chain compound are quickly completely obtained using molecular sieve centrifugal column Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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Abstract
The present invention provides a kind of method and its application that mRNA ribosomes new polypeptide chain compound is quickly completely obtained using molecular sieve centrifugal column.By the present invention in that cracking biological sample with lysate, pyrolysis product is obtained；The fragment in lysate is removed by differential centrifugation, the supernatant after being cracked；Supernatant after purifying cracking using molecular sieve centrifugal column, obtains the mRNA ribosomes new polypeptide chain compound of high-purity.This method does not need expensive instrument ultracentrifuge, and easy to operate, step is few, the probability polluted by RNase is small, experimental repeatability is high, and molecular sieve centrifugation column purification step time-consuming only needs 10min or so, highly shortened experimental period, and it can in high volume operate, solving sucrose density supercentrifugation, time-consuming, and operation difficulty is high, and flux is low to wait limitation, the RNC-mRNA sequencing data that high quality can be obtained simultaneously, has important impetus to RNC Technique Popularizing.
Description
Technical field
The invention belongs to technical field of life science, in particular to a kind of quickly completely to be obtained using molecular sieve centrifugal column
The method and its application of mRNA ribosomes new polypeptide chain compound.
Background technique
Translational control is the intracorporal important regulating and controlling level of life, and translational control occupies 50% or more that life entity always regulates and controls,
Gene, which is expressed, in life entity does not represent into translation process synthetic proteins matter, therefore grinds to the translation situation of life entity
Study carefully particularly important.When cell is translated, ribosomes is incorporated on mRNA chain and mobile and synthetic proteins matter, this complete mRNA
The complex formed with ribosomes and new polypeptide chain is exactly mRNA ribosomes new polypeptide chain compound (Ribosome
Nascent-chain Complex, RNC), complete undegradable RNC is only extracted, the mRNA translated could be carried out
Qualitative, quantitative research.
The conventional extraction of RNC is sucrose density supercentrifugation at present, and the method can extract complete undegraded
RNC, retain ribosomes on overall length mRNA and new polypeptide chain for thereafter various biochemistry and molecular biologies study (Wang
et al.,2013；Zhang et al., 2009), can be used for animal, plant, the culture cell of microorganism and tissue (middle promulgated by the State Council
Bright patent ZL 201510332732, ZL 201410452522, ZL 201410387213).The document and patent delivered at present
What is used is all sucrose density supercentrifugation, although sucrose density supercentrifugation can extract complete undegradable RNC,
But this method needs expensive instrument such as ultracentrifuge, consumptive material (such as ultracentrifugation pipe) is at high cost, cumbersome including super
Fast centrifuge pre-cooling 2h, the preparation of Sucrose gradient solutions, trim (it is required that error is within 0.01g) before machine on ultracentrifuge,
The resuspension etc. of RNC in ultracentrifugation pipe, thus experimental period it is very long (different according to sample size and rotary head need 5~13h, if
Blindly shortening centrifugation time by improving centrifugal force will lead to impurity pollution), sucrose solution can not sterilize thus be mixed into RNase
Probability it is big, trim process need by cracking supernatant move to ambient operation, easily cause the degradation of RNC, the rate of recovery is lower, between batch
Careless manipulation be easy to cause difference, and the experience and operation fine degree to operator have very big requirement.The extraction of micro-example
Very risky, precipitating naked eyes are difficult to find and be easy dissolution, and the sample Limited Number extracted every time, these restrictive condition poles
The big laboratory for hindering RNC technology is promoted and business application.
Traditional tomographic system (such as GE AKTA Pure system) supports the RNC in purifying biological sample in principle, still
Since tomographic system purification time is too long, many experiment components can not high pressure inactivation RNase, especially chromatographic column and filler can not
Inactivate RNase, easily RNC-RNA is caused to degrade, full length mRNA cannot be got, also just can not to the mRNA translated into
The research of row qualitative, quantitative is only used for extracting ribosomal protein.RNC disintegration is even resulted in when degrading serious, new polypeptide chain is all
It is difficult to extract.
Therefore the maturation method for extracting RNC at present only has sucrose density supercentrifugation, lacks a kind of quick, easy, high
The technology of flux.
Summary of the invention
The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide a kind of use molecular sieve centrifugal column
The quickly complete method for obtaining mRNA ribosomes new polypeptide chain compound.Present invention firstly provides use molecular sieve centrifugal column method
MRNA ribosomes new polypeptide chain compound is extracted, technological package scheme is optimized.It is straight using molecular sieve supramolecular quantity of material
The characteristic penetrated is connect, in conjunction with low-speed centrifugal as means of purification, does not need expensive instrument ultracentrifuge and tomographic system, is only needed
Want common low speed centrifuge, experimental procedure is few, easy to operate, small by RNase contamination probability, substantially shorten experimental period,
It can be obtained while greatly reducing experimental cost and sucrose density supercentrifugation is identical in quality, even more preferably RNC-mRNA
Sequencing data can effectively overcome current method at high cost, complicated for operation, the low technological deficiency of flux.
Another object of the present invention is to provide above-mentioned use molecular sieve centrifugal column, quickly completely acquisition mRNA ribosomes is new
The application of the method for raw peptide chain cpd.
The purpose of the invention is achieved by the following technical solution: a kind of quickly completely to obtain mRNA using molecular sieve centrifugal column
The method of ribosomes new polypeptide chain compound, includes the following steps:
(1) biological sample is cracked using lysate, obtains pyrolysis product；It is removed by differential centrifugation broken in lysate
Piece, the supernatant after being cracked；
(2) supernatant after purifying cracking using molecular sieve centrifugal column, obtains the mRNA ribosomes nascent peptide of high-purity
Chain cpd.
Lysate described in step (1) is the salt buffer for extending inhibitor and Triton X-100 containing translation.Step
(1) commercial product can be used in the lysate described in, using common cell lysing methods, art technology disclosed in the prior art
Personnel can select method and condition appropriate according to the actual situation, and the present invention is not especially limited this.
It is preferably cycloheximide or chloramphenicol that translation in the lysate, which extends inhibitor,；Cycloheximide is suitable for true
Core biology, chloramphenicol are suitable for prokaryotes.
The concentration of the cycloheximide is preferably 0.05~0.15mg/mL；More preferably 0.1mg/mL.
The concentration of the chloramphenicol is preferably 0.05~0.15mg/mL；More preferably 0.1mg/mL.
The concentration of the Triton X-100 is percent by volume 0.1~1.5%；More preferably 0.2~1%.
Biological sample described in step (1) includes eucaryote and prokaryotes.
When the biological sample is eucaryote, the composition of the lysate is preferably as follows: 15~25mM, pH7.4
HEPES-KOH solution, 10~20mM MgCl2, 150~250mM KCl, 50~150 μ g/ml cycloheximides, 1~3mM
DTT, 0.5~1.5% (v/v) Triton X-100；It is more preferably as follows: HEPES-KOH solution, the 15mM of 20mM, pH7.4
MgCl2, 200mM KCl, 100 μ g/ml cycloheximides, 2mM DTT, 1% (v/v) Triton X-100.
When the biological sample is prokaryotes, the composition of the lysate is preferably as follows: 5~15mM, pH7.8
Tris-HCl, 5~15mM MgCl2, 30~70mM NH4Cl, 0.1~0.3% (v/v) TritonX-100,100 μ g/ml chlorine are mould
Element；It is more preferably as follows: Tris-HCl, 10mM MgCl of 10mM, pH7.82、50mM NH4Cl, 0.2% (v/v) TritonX-
100,100 μ g/ml chloramphenicol.
In order to be more advantageous to from the complete RNC of extraction from biological material, different biological samples is split in addition
Processing before solution liquid can refer to national inventing patent ZL 201510332732, ZL 201410452522, ZL
201410387213。
When the biological sample is prokaryotes, it is also preferable to include following processing steps before the lysate is added
It is rapid: lysozyme soln and MgCl being added in prokaryote2Solution processing.The step is conducive to prokaryote and is split
Liquid cracking is solved, while keeping the integrality of RNC.
The condition of differential centrifugation described in step (1) is preferably centrifuged 10~20min in 16000~18000g；It is more excellent
It selects and is centrifuged 15min in 17000g.
Molecular sieve used in molecular sieve centrifugal column described in step (2) be centrifuged the preferred exclusion range of column packing be 2 ×
104~8 × 106The molecular sieve of Da is centrifuged column packing.
Specific steps described in step (2) using the supernatant after molecular sieve centrifugation column purification cracking are preferably as follows:
(A) take out molecular sieve centrifugal column, using column balance buffering liquid carry out column equilibration, be subsequently placed under 2~8 DEG C of environment to
Column packing sedimentation is complete；
(B) the molecular sieve centrifugal column settled, the solution being centrifuged off in column, the pillar prepared are taken out；
(C) supernatant after cracking is added drop-wise to molecular sieve centrifugal column column bed surface, filtrate is collected by centrifugation, obtains purifying
High-purity RNC.
Molecular sieve centrifugal column described in step (A) is including but not limited to MicroSpin S-400 HR-GE
Healthcare molecular sieve centrifugal column.It can reach using other manufacturer's molecular sieve centrifugal columns of close exclusion range identical pure
Change effect.
The composition of column balance buffering liquid described in step (A) is as follows:
When the biological sample is eucaryote, the composition of the column balance buffering liquid is preferably as follows: 15~25mM,
HEPES-KOH solution, the 10~20mM MgCl of pH7.42, 150~250mM KCl；It is more preferably as follows: 20mM, pH7.4's
HEPES-KOH solution, 15mM MgCl2,200mM KCl；
When the biological sample is prokaryotes, the composition of the column balance buffering liquid is preferably as follows: 5~15mM,
Tris-HCl, the 5~15mM MgCl of pH7.82, 30~70mM NH4Cl；It is more preferably as follows: the Tris-HCl of 10mM, pH7.8,
10mM MgCl2、50mM NH4Cl。
Sedimentation described in step (A) refers to 2~8 DEG C of gravitational settlings 6 hours or more completely；It is more preferably heavy in 4 DEG C of gravity
8~16h drops.
The condition of centrifugation described in step (B) is preferably centrifuged 1~3min in 500~700g；More preferably in 600g
It is centrifuged 2min.
The condition of centrifugation described in step (C) is preferably centrifuged 30~90s in 500~700g；More preferably in 600g
It is centrifuged 60s.
The above-mentioned method that mRNA ribosomes new polypeptide chain compound is quickly completely obtained using molecular sieve centrifugal column is existed
MRNA builds the application in the sequencing of library, preferably includes following steps: the RNC that the above method obtains being extracted, RNC- is obtained
Then RNA carries out mRNA and builds library sequencing.
The extraction can be realized by using Trizol or kit；It is tried it is preferred that being extracted by using TRIzol RNA
Agent.
The present invention has the following advantages and effects with respect to the prior art:
For sucrose density supercentrifugation because to use ultracentrifuge as laboratory apparatus, experimental procedure is more, and operation is difficult
Degree is high, and centrifugation time is very long.Sucrose solution because can not high pressure can introduce RNase pollution, ultracentrifugation pipe trim consumes before upper machine
Duration easily causes the degradation of RNC, so that fubaritic arrive full length mRNA.The method of the present invention substitutes sugarcane using molecular sieve centrifugal column
Carbohydrate density supercentrifugation is extracted mRNA ribosomes new polypeptide chain compound and is compared with sucrose density supercentrifugation: not needing
Expensive instrument ultracentrifuge, easy to operate, step is few, and the probability polluted by RNase is small, and experimental repeatability is high, molecular sieve from
Stem purification step time-consuming only needs 10min or so, greatly shortens experimental period, and can in high volume operate.It solves and works as
Time-consuming for front method, and operation difficulty is high, the low limitation of flux, while can obtain the RNC-mRNA sequencing data of high quality, allows
Common lab also can be carried out RNC research, and company is capable of providing quickly large batch of RNC sequencing commerce services, pushes away to RNC technology
Extensively there is important impetus.
Detailed description of the invention
Fig. 1 is that the RNC-RNA that the different preparation methods of embodiment 1 obtain is detected by 2% agarose gel electrophoresis
Result figure；Wherein, swimming lane 1 is DNA Marker, and swimming lane 2 is mouse kidney RNA (extraction of Trizol method), and swimming lane 3 is cracking supernatant
Liquid RNA (extraction of Trizol method), swimming lane 4 and swimming lane 5 are that molecular sieve centrifugal column extracts to obtain RNC-RNA, and swimming lane 6 and swimming lane 7 are
The RNC-RNA that sucrose density supercentrifugation is extracted.
Fig. 2 is the RNC that the conventional gel chromatography method that embodiment 1 obtains extracts, and swimming lane 1 is DNA Marker, swimming lane 2-3
For the RNC-RNA for using conventional gel chromatography method to extract.
Fig. 3 is the DNase I gene mRNA coverage diagram spectrogram that embodiment 1 obtains, the corresponding molecular sieve centrifugal column of curve 1 in figure
Method, the corresponding sucrose density supercentrifugation of curve 2.
Fig. 4 is the molecular sieve chromatography method and sucrose density supercentrifugation gene expression amount correlation that embodiment 1 obtains
Analyze result figure.
Fig. 5 is the molecular sieve centrifugal column method and technology duplicate factor expression quantity correlation analysis result figure that embodiment 1 obtains.
Fig. 6 is the sucrose density supercentrifugation and technology duplicate factor expression quantity correlation analysis knot that embodiment 1 obtains
Fruit figure.
Fig. 7 is that the RNC-RNA that the different preparation methods of embodiment 2 obtain is detected by 3% agarose gel electrophoresis
Result figure；Wherein, swimming lane 1 is DNA Marker, and swimming lane 2 is A549 cell total rna (extraction of Trizol method), swimming lane 3-6 difference
For the RNC-RNA that molecular sieve centrifugal column method is extracted, swimming lane 7 is the RNC-RNA that sucrose density supercentrifugation is extracted.
Fig. 8 is the Rps2 gene mRNA coverage diagram spectrogram that embodiment 2 obtains, the corresponding molecular sieve centrifugal column method of curve 1 in figure,
Curve 2 corresponds to sucrose density supercentrifugation.
Fig. 9 is the molecular sieve chromatography method and sucrose density supercentrifugation gene expression amount correlation that embodiment 2 obtains
Analyze result figure.
Figure 10 is the molecular sieve centrifugal column method and technology duplicate factor expression quantity correlation analysis result that embodiment 2 obtains
Figure.
Figure 11 is that the RNC-RNA that the different preparation methods of embodiment 3 obtain is detected by 2% agarose gel electrophoresis
Result figure: where swimming lane 1 is DNA Marker, and swimming lane 2 is Escherichia coli total serum IgE (extraction of Trizol method), and swimming lane 3 is cracking
Product supernatant RNA (extraction of Trizol method), swimming lane 4-6 are the RNC-RNA that molecular sieve centrifugal column method is extracted, and swimming lane 7 is that sucrose is close
Spend the RNC-RNA that supercentrifugation is extracted.
Figure 12 is the RpmB gene mRNA coverage diagram spectrogram that embodiment 3 obtains, the corresponding molecular sieve centrifugal column of curve 1 in figure
Method, the corresponding sucrose density supercentrifugation of curve 2.
Figure 13 is the molecular sieve chromatography method and sucrose density supercentrifugation gene expression amount correlation that embodiment 3 obtains
Analyze result figure.
Figure 14 is the molecular sieve centrifugal column method and technology duplicate factor expression quantity correlation analysis result that embodiment 2 obtains
Figure.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Unless otherwise defined, the present invention used in all scientific and technical terms have with the present invention relates to technologies to lead
The normally understood identical meaning of the technical staff in domain, such as " molecular sieve centrifugal column " and " gel filtration centrifugal column " " exclusion chromatography
Centrifugal column " " gel chromatography centrifugal column ".
Heretofore described molecular sieve centrifugal column is not equal to gel chromatography column or molecular sieve chromatography, in means of purification
It is all different in equipment.It usually requires to be purified in tomographic system using gel chromatography column purification, such as GE AKTA
Pure system, individual column is difficult to operate, impracticable.
Heretofore described quickly completely obtains mRNA ribosomes new polypeptide chain compound using molecular sieve centrifugal column
The core implementation tool of method is molecular sieve centrifugal column.
Embodiment 1
It is newborn that animal tissue (BALB/C mice kidney) interior mRNA ribosomes is quickly completely obtained using molecular sieve centrifugal column
Peptide chain cpd, the specific steps are as follows:
(1) molecular sieve centrifugal column (MicroSpin S-400 HR-GE Healthcare) is taken out, using 3ml without RNase
Column balance buffering liquid (HEPES-KOH solution, the 15mM MgCl of 20mM, pH7.42, 200mM KCl) carry out column equilibration, then
Enter 4 DEG C of refrigerators and waits for column packing sedimentation overnight.
(2) 6 week old BALB/C mices (Guangdong Province medical animal experiment center) is dissected, obtains kidney organ, uses DPBS
Solution Rapid Cleaning blood, is put into liquid nitrogen and suddenly freezes.
(3) mouse kidney is put into grinding pipe, is ground using tissue grinder instrument.
(4) cell pyrolysis liquid of 1ml is added into grinding pipe, the composition of cell pyrolysis liquid is as follows: 20mM, pH7.4's
HEPES-KOH solution, 15mM MgCl2, 200mM KCl, 100 μ g/ml cycloheximide, 2mM DTT (dithiothreitol (DTT)), 1%
(v/v)Triton X-100；25min is cracked, mouse kidney tissue pyrolysis product is obtained.
(5) mouse kidney tissue pyrolysis product is centrifuged 5min in 17000g, takes supernatant, removes big fragment；It will obtain
Supernatant then at 17000g be centrifuged 15min, remove fractionlet, obtained supernatant is transferred in another EP pipe.
(6) the molecular sieve centrifugal column that step (1) has settled is taken out, the liquid in column is removed in 600g centrifugation 2min, obtains
Prepare the molecular sieve centrifugal column completed.
(7) the cracking supernatant for quickly 50 μ l steps (5) being taken finally to obtain, careful dropwise are added to molecular sieve centrifugal column column
Bed surface, not break up column bed surface, be centrifuged 1min in 600g, collect filtrate, filtrate, that is, purified RNC.(8) it uses
TRIzol RNA extracts reagent (Invitrogen company), operates by its instruction manual method, extracts from above-mentioned RNC solution
RNC-RNA。
(9) sucrose is used from batch mouse kidney tissue by 1 the method for embodiment in patent ZL 201410452522
Density supercentrifugation extracts RNC, and extracts RNC-RNA with Trizol reagent, as a comparison.
(10) the cracking supernatant in step (5) is taken, extracts RNC, Sephacryl on supernatant using gel chromatography system
S400 chromatographic column (GE Healthcare), with 1 × RBbuffer (HEPES-KOH solution, 15mM MgCl of 20mM, pH7.42、
The cycloheximide of 200mM KCl, 100 μ g/ml, 2mM DTT (dithiothreitol (DTT))) elution, it picks up and penetrates peak, use TRIzol
RNA extracts reagent (Invitrogen), operates by its instruction manual method, extracts RNC-RNA from above-mentioned RNC solution.
(11) using the library MGIEasyRNA reagent preparation be set with to use molecular sieve centrifugal column and sucrose density exceed the speed limit from
The mRNA in RNC-RNA that heart method is extracted carries out sequencing and builds library, and BGISEQ-500 sequenator carries out the sequencing of two generations, obtains translation group
Sequencing information (the mRNA quantitative information translated).
(12) to examine this method that can propose high quality RNC same as sucrose density supercentrifugation, this experiment is used
Carry out sucrose density ultracentrifugation with a batch of tissue sample and extract RNC, then with the RNC that uses molecular sieve centrifugal column to extract
Carry out experimental result comparative analysis.
(13) to examine the method for the present invention that can propose undegradable RNC-RNA from tissue, we are by the tissue of part
RNC-RNA sample carry out 2% agarose gel electrophoresis detect its integrality, with sucrose density supercentrifugation extract RNC into
Row compares, and is dyed using GelRed, ultraviolet gel imaging system imaging.As a result swimming lane 4,5 is molecular sieve centrifugation as shown in Figure 1:
Column extracts RNC-RNA, and swimming lane 6,7 is that sucrose density supercentrifugation extracts RNC-RNA.As shown in Figure 1, molecular sieve centrifugal column mentions
28s rRNA and 18s the rRNA band of the RNC-RNA obtained is limpid in sight, without obvious hangover, no degradation, in RNC integrity degree
Upper and sucrose density supercentrifugation has same extraction effect, the RNC-RNA's that sucrose density supercentrifugation obtains
Visually visible above 28S rRNA band there are miscellaneous band pollutions, and molecular sieve centrifugal column method is not present above 28S rRNA band
Miscellaneous band.The RNC component that Fig. 2 is extracted using conventional gel chromatography method (using GE AKTA system), RNA degradation is serious, 28S
It is degradable with 18S rRNA, it is not useable for mRNA sequencing.Therefore electrophoresis result shows that molecular sieve centrifugal column purification effect is better than
Sucrose density supercentrifugation and conventional gel chromatography.
(14) whole sequencing fragments (reads) of sequencing data are subjected to connector removal, using FANSe3 algorithm compare to
Mouse transcription group reference sequences and mouse reference genome.
(15) to examine the method for the present invention that can extract full length mRNA, the transcript profile reference sequences in step (14) are used
Comparison data draws full length mRNA and covers map, by taking gene DNase I as an example.As a result as shown in figure 3, sequencing result and sucrose are close
It is identical to spend supercentrifugation, whole mRNA can be covered.
(16) for examine the RNC-mRNA quantitative gene expression effect that extracts of the method for the present invention and sucrose density exceed the speed limit from
Heart method similitude is high, calculates RPKM using the transcript profile reference sequences comparison data in step (14), calculates microbiological specimens
The Pearson phase of molecular sieve centrifugal column RNC gene expression amount RPKM and sucrose density supercentrifugation RNC gene expression amount RPKM
Relationship number, as a result such as Fig. 4.By Fig. 4 result it is found that molecular sieve centrifugal column RNC gene expression amount RPKM and sucrose density exceed the speed limit from
The Pearson correlation coefficient R of heart method RNC gene expression amount RPKM2=0.98408, correlation is very high, it is believed that repeatability is very
It is good, show molecular sieve centrifugal column and sucrose density supercentrifugation to tissue RNC extraction effect having the same.
(17) to examine technology repeatability of the invention, the gene expression amount RPKM data in (16) is used to calculate separately point
Son sieve centrifugal column method and sucrose density supercentrifugation and the duplicate Pearson correlation coefficient R of its technology2, as a result as shown,
Fig. 5 is that molecular sieve centrifugal column method and technology compute repeatedly as a result, R2=0.99229, Fig. 6 are sucrose density supercentrifugation and skill
Art computes repeatedly as a result, R2=0.98239.The result shows that: the technology repeatability of molecular sieve centrifugal column method is super better than sucrose density
Fast centrifugal process.
(18) to examine identified for genes quantity of the invention similar with sucrose density supercentrifugation, using in step (14)
Transcript profile reference sequences comparison data statistics compare to the sequencing fragment quantity (readcounts) on each gene,
The gene of readcounts > 10 is considered the gene for having expression in the sample, and the results are shown in Table 1.As seen from the results in Table 1,
The RNC that molecular sieve centrifugal column extracts can be quantified to 14567 genes, and sucrose density supercentrifugation can be arrived quantitatively
14648 genes, the fluctuation of identified for genes quantity, if purification effect is poor, identify within the scope of normal technology recurrent fluctuation
Gene number can significantly increase.
(19) RNC- extracted for the RNC-mRNA and sucrose density supercentrifugation that examine the method for the present invention to extract
Excessive differential gene is not present in mRNA, using readcounts data in edgeR analytical procedure (18), identifies molecular sieve centrifugation
The RNC that column extracts and sucrose density supercentrifugation extract the differential gene between RNC, and the gene of PValue < 0.01 is considered
Differential gene, the results are shown in Table 2.Differential gene is not present between molecular sieve centrifugal column and sucrose density supercentrifugation, it can
To think two technologies without significant difference.
Embodiment 2
Quickly completely Non-small cell lung carcinoma cell A549 (Cell Bank of Chinese Academy of Sciences) is obtained using molecular sieve centrifugal column
Interior mRNA ribosomes new polypeptide chain compound, steps are as follows:
(1) molecular sieve centrifugal column (MicroSpin S-400 HR-GE Healthcare) is taken out, uses the column equilibration of 3ml
Buffer (HEPES-KOH solution, the 15mM MgCl of 20mM, pH7.42, 200mM KCl) carry out column equilibration, be subsequently placed in 4 DEG C
Refrigerator waits for column packing sedimentation overnight.
(2) 1ml cell pyrolysis liquid, the composition of cell pyrolysis liquid are added into Tissue Culture Flask (10,000,000 A549 cells)
It is as follows: HEPES-KOH solution, the 15mM MgCl of 20mM, pH7.42, 200mM KCl, 100 μ g/ml cycloheximides, 2mM DTT,
1% (v/v) Triton X-100；25min is cracked, product of cell lysis is obtained.
(3) product of cell lysis removes big fragment in 17000g centrifugation 5min, takes supernatant 17000g centrifugation 15min removal
Then cracking supernatant is transferred in another EP pipe by fractionlet.
(4) the molecular sieve centrifugal column settled is taken out, the liquid in column is removed in 600g centrifugation 2min, obtains preparation and complete
Molecular sieve centrifugal column.
(5) 50 μ l are quickly taken to crack supernatant, careful dropwise is added to molecular sieve centrifugal column column bed surface, not break up column
Filtrate, filtrate, that is, purified RNC are collected in 600g centrifugation 1min in bed surface.
(6) reagent (Invitrogen company) is extracted using TRIzol RNA, by the operation of its instruction manual method, from above-mentioned
RNC-RNA is extracted in RNC solution.
(7) it uses sucrose density supercentrifugation to extract RNC as control, remaining cracking supernatant is added separately to
Different (is buffered equipped with 14.5mL30% (w/v) sucrose solution surface by the column equilibration of 100 μ g/ml cycloheximides and 2mM DTT
Liquid is prepared), precipitating is resuspended by 4 DEG C, 42500rpm ultracentrifugation 5h, and whole process is operated on ice.It is mentioned using TRIzol RNA
Reagent (Invitrogen company) is taken, is operated by its instruction manual method, RNC-RNA is extracted from RNC.
(8) using MGIEasy RNA library reagent preparation be set with to use molecular sieve centrifugal column and sucrose density exceed the speed limit from
The mRNA in RNC-RNA that heart method is extracted carries out sequencing and builds library, and BGISeq-500 sequenator carries out the sequencing of two generations, obtains translation group
Sequencing information (the mRNA quantitative information translated).
(9) to examine this method that can propose high quality RNC same as sucrose density supercentrifugation, this experiment uses same
A batch of cell sample carries out sucrose density ultracentrifugation and extracts RNC, then with the RNC that uses molecular sieve centrifugal column to extract into
Row experimental result comparative analysis.
(10) to examine the method for the present invention that can propose undegradable RNC-RNA from cell, we are by the cell of part
RNC-RNA sample carry out 2% agarose gel electrophoresis detect its integrality, with sucrose density supercentrifugation extract RNC into
Row compares, and is dyed using GelRed, ultraviolet gel imaging system image checking.As a result as shown in fig. 7, swimming lane 3,4,5,6 is point
The RNC-RNA that son sieve centrifugal column method is extracted, swimming lane 7 are the RNC-RNA that sucrose density supercentrifugation is extracted.As shown in Figure 7, divide
28s rRNA and 18s the rRNA band for the RNC-RNA that son sieve centrifugal column extracts is limpid in sight, and no obvious degradation is complete in RNC
On degree and sucrose density supercentrifugation has same extraction effect, in the 28S rRNA band of sucrose density supercentrifugation
Top naked eyes it is visible there are miscellaneous band pollution and molecular sieve centrifugal column method above 28S rRNA band be not present miscellaneous band, electrophoresis result
Show that this method purification effect is better than sucrose density supercentrifugation.
(11) whole sequencing fragments (reads) of sequencing data are subjected to connector removal, using FANSe3 algorithm compare to
People's transcript profile reference sequences and ginseng examine genome sequence.
(12) to examine the method for the present invention that can extract full length mRNA, the transcript profile reference sequences in step (11) are used
Comparison data draws full length mRNA and covers map, by taking gene Rps2 as an example, as a result as shown in figure 8, sequencing result and sucrose density
Supercentrifugation is identical, can cover whole mRNA.
(13) the cell RNC-mRNA quantitative gene expression effect and sucrose density to examine the method for the present invention to extract surpass
Fast centrifugal process is similar, calculates RPKM using the transcript profile reference sequences comparison data in step (11), amounts to calculation microbiological specimens
Gel filtration RNC gene expression amount RPKM and sucrose density supercentrifugation RNC gene expression amount RPKM Pearson
Related coefficient.As a result as shown in figure 9, molecular sieve centrifugal column RNC gene expression amount RPKM and sucrose density supercentrifugation RNC base
Because of the Pearson correlation coefficient square R of expression quantity RPKM2=0.98239, correlation is very high, it is believed that the two is technology weight
It is multiple, show molecular sieve centrifugal column and sucrose density supercentrifugation to cell RNC extraction effect having the same.
(14) to examine technology repeatability of the invention, the gene expression amount RPKM data in (13) is used to calculate molecular sieve
Centrifugal column method and the duplicate Pearson correlation coefficient R of its technology2, the results are shown in Figure 10, Figure 10 be molecular sieve centrifugal column method with
Technology computes repeatedly as a result, R2=0.99363.The result shows that: molecular sieve centrifugal column method has good technology repeatability.
(15) to examine the RNC identified for genes quantity of the invention extracted similar with sucrose density supercentrifugation, step is used
Suddenly the transcript profile reference sequences comparison data statistics in (11) is compared to the sequencing fragment number on each gene
(readcounts), the gene of readcounts > 10 is considered the gene for having expression in the sample, and the results are shown in Table 1.By
1 result of table is it is found that the RNC that molecular sieve centrifugal column extracts can be quantified to 14704 genes, and sucrose density supercentrifugation energy
Enough quantitative to 14736 genes, the fluctuation of identified for genes quantity is within the scope of normal technology recurrent fluctuation.
(16) RNC- extracted for the RNC-mRNA and sucrose density supercentrifugation that examine the method for the present invention to extract
Excessive differential gene is not present in mRNA, using readcounts data in edgeR analytical procedure (15), identifies molecular sieve centrifugation
The RNC that column extracts and sucrose density supercentrifugation extract the differential gene between RNC, and the gene of PValue < 0.01 is considered
Differential gene, the results are shown in Table 2.As shown in Table 2,3 are only existed between molecular sieve centrifugal column and sucrose density supercentrifugation
A differential gene, it is believed that two technologies belong within the scope of sequencing error without significant difference.
Embodiment 3
Microorganism (e. coli k-12 BW25113, BioVector China is quickly completely obtained using molecular sieve centrifugal column
Plasmid vector strain cell protein antibodies gene collection) interior mRNA ribosomes new polypeptide chain compound, steps are as follows:
(1) molecular sieve centrifugal column (MicroSpin S-400 HR-GE Healthcare) is taken out, uses the column equilibration of 3ml
Buffer (Tris, 50mM NH of 10mM, pH7.84Cl、0.01M MgCl2) column equilibration is carried out, then enter 4 DEG C of refrigerators and waits for that column is filled out
Material sedimentation is overnight.
(2) by Escherichia coli BW25113 glycerol kind scribing line LB plate, overnight incubation.
(3) choose single colonie on plate, be inoculated in 3mlLB fluid nutrient medium, 37 DEG C, 200rpm activation overnight.
(4) by the seed liquor after the inoculum concentration inoculation activation of percent by volume 1%, it is inoculated in 50ml LB liquid medium
In, 37 DEG C, 200rpm cultivates to culture solution OD600=0.6.
(5) chloramphenicol is added into culture solution obtained by step (4), concentration of the chloramphenicol in culture is 100 μ g/ml, ice
5min quickly cooling is shaken in water to 4 DEG C, 4 DEG C, 5000g centrifugation 5min remove supernatant.
(6) the 50ml PBS (containing 100 μ g/ml chloramphenicol) of 4 DEG C of pre-coolings is added to precipitating obtained by step (5), precipitating is resuspended,
4 DEG C, 5000g centrifugation 5min collection bacterium, abandon supernatant.
(7) step (6) are repeated；
(8) 4 DEG C of pre-cooling lysozyme (lysozyme) buffer (Sigma) 5.4ml, weight is added to precipitating obtained by step (7)
Outstanding cell precipitation, is added 0.6ml lysozyme enzyme solution (12.5mg/ml, lysozyme are dissolved in PBS solution), and gently piping and druming mixes, and is placed in
5min is stood on ice, is gently shaken up every 1min, and the MgCl that concentration is 1M is added2Solution 0.15ml, gently shakes up, 4 DEG C,
5000g is centrifuged 5min and receives bacterium, abandons supernatant.
(9) upwards step gained precipitating be added 0.6ml4 DEG C pre-cooling cell pyrolysis liquid (Tris-HCl of 10mM, pH7.8,
50mM NH4Cl、10mM MgCl2, 0.2% (v/v) TritionX-100,100 μ g/ml chloramphenicol), piping and druming be resuspended precipitating, on ice
5min is stood, then cracking supernatant is transferred in another EP pipe by 4 DEG C, 17000g centrifugation 15min.
(10) the molecular sieve centrifugal column settled is taken out, the liquid in column is removed in 600g centrifugation 2min, obtains having prepared
At molecular sieve centrifugal column.
(11) 50 μ l are quickly taken to crack supernatant, careful dropwise is added to molecular sieve centrifugal column column bed surface, not break up
Filtrate, filtrate, that is, purified RNC are collected in 600g centrifugation 1min in column bed surface.
(12) reagent (Invitrogen company) is extracted using TRIzol RNA, by the operation of its instruction manual method, from upper
It states in RNC solution and extracts RNC-RNA.
(13) sucrose density supercentrifugation is used to extract RNC as control.Remaining cracking supernatant is separately added into
Onto the different HEPE buffers equipped with 14.5mL35% (w/v) sucrose concentration, 4 DEG C of 42500rpm ultracentrifugation 5h will sink
It forms sediment and is resuspended, whole process is operated on ice.Reagent (Invitrogen company) is extracted using TRIzol RNA, by its instruction manual
Method operation, extracts RNC-RNA from RNC.
(14) molecular sieve centrifugal column and sucrose are used to microbiological specimens using MGIEasy RNA library reagent preparation suit
The mRNA in RNC-RNA that density supercentrifugation is extracted carries out sequencing and builds library, and BGI Seq-500 sequenator carries out the survey of two generations
Sequence obtains translation group sequencing information (the mRNA quantitative information translated).
(15) to examine this method that can propose high quality RNC same as sucrose density supercentrifugation, this experiment is used
Sucrose density ultracentrifugation is carried out with a batch of microbiological specimens and extracts RNC, is then extracted with molecular sieve centrifugal column is used
RNC carries out experimental result comparative analysis.
(16) to examine the method for the present invention that can propose undegradable RNC-RNA from microorganism, we are by the micro- life in part
Object RNC-RNA sample carries out 2% agarose gel electrophoresis and detects its integrality, the RNC extracted with sucrose density supercentrifugation
It is compared, is dyed using GelRed, ultraviolet gel imaging system imaging shooting, as a result as shown in figure 11, swimming lane 4,5,6 is point
The RNC-RNA that son sieve centrifugal column extracts, swimming lane 7 are the RNC-RNA that sucrose density supercentrifugation is extracted.As shown in Figure 11, divide
23s rRNA and 16s the rRNA band for the RNC-RNA that son sieve centrifugal column extracts is limpid in sight, no obvious degradation and sucrose density
Supercentrifugation, which is compared, has same extraction effect.
(17) whole sequencing fragments (reads) of sequencing data are subjected to connector removal, using FANSe3 algorithm compare to
Genome of E.coli and Escherichia coli transcript profile.
(18) to examine the method for the present invention that can extract full length mRNA, the transcript profile reference sequences in step (17) are used
Comparison data draws full length mRNA and covers map, by taking gene RpmB as an example, as a result as shown in figure 12, sequencing result and sucrose density
Supercentrifugation is identical, can cover whole mRNA.
(19) for examine the RNC-mRNA quantitative gene expression result that extracts of the method for the present invention and sucrose density exceed the speed limit from
Heart method is identical, calculates RPKM using the transcript profile reference sequences comparison data in step (17), calculates the molecule of microbiological specimens
It is related to the Pearson of gene expression amount RPKM of sucrose density supercentrifugation RNC to sieve centrifugal column RNC gene expression amount RPKM
Coefficient.As a result as shown in figure 13, molecular sieve centrifugal column method RNC gene expression amount RPKM and sucrose density supercentrifugation RNC base
Because of the Pearson correlation coefficient square R of expression quantity RPKM2=0.98627, correlation is very high, it is believed that the two is technology weight
It is multiple.
(20) to examine technology repeatability of the invention, the gene expression amount RPKM data in (19) is used to calculate molecular sieve
Centrifugal column method and the duplicate Pearson correlation coefficient R of its technology2, as a result as shown in figure 14, Figure 14 be molecular sieve centrifugal column method with
Technology computes repeatedly as a result, R2=0.98329.The result shows that: molecular sieve centrifugal column method has good technology repeatability.
(21) to examine extraction RNC-mRNA identified for genes quantity of the invention similar with sucrose density supercentrifugation, make
It is compared with the transcript profile reference sequences comparison data statistics in step (17) to the sequencing fragment number on each gene
(readcounts), the gene of readcounts > 10 is considered the gene for having expression in the sample, and the results are shown in Table 1.By
As a result it is found that the RNC that molecular sieve centrifugal column extracts can be quantified to 4189 genes, and sucrose density supercentrifugation can determine
It measures to 4118 genes, since, there are sequencing throughput difference, the fluctuation of identified for genes quantity is repeated in normal technology between sample
In fluctuation range.
(22) RNC- extracted for the RNC-mRNA and sucrose density supercentrifugation that examine the method for the present invention to extract
Excessive differential gene is not present in mRNA, uses readcounts data in edgeR analytical procedure (21), analyzing molecules sieve centrifugation
The RNC that column extracts and sucrose density supercentrifugation extract the differential gene between RNC, and the gene of PValue < 0.01 is considered
Differential gene, the results are shown in Table 2.As shown in Table 2, it is not present between molecular sieve centrifugal column method and sucrose density supercentrifugation
Differential gene, it is believed that significant difference is not present in two technologies, belongs within the scope of sequencing error.
The identified for genes quantity of 1 embodiment 1,2,3 of table
The differential gene quantity of 2 embodiment 1,2,3 of table identification
| Sample | Differential gene number |
| BALB/C mice kidney | 0 |
| Cell (A549) | 3 |
| E. coli k-12 BW25113 | 0 |
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of method for quickly completely being obtained mRNA ribosomes new polypeptide chain compound using molecular sieve centrifugal column, feature are existed
In including the following steps:
(1) biological sample is cracked using lysate, obtains pyrolysis product；The fragment in lysate is removed by differential centrifugation, is obtained
Supernatant after to cracking；
(2) supernatant after purifying cracking using molecular sieve centrifugal column, the mRNA ribosomes new polypeptide chain for obtaining high-purity are multiple
Close object.
2. according to claim 1 quickly completely obtain mRNA ribosomes new polypeptide chain compound using molecular sieve centrifugal column
Method, it is characterised in that:
Lysate described in step (1) is the salt buffer for extending inhibitor and Triton X-100 containing translation；
The condition of differential centrifugation described in step (1) is to be centrifuged 10~20min in 16000~18000g；
The exclusion range that molecular sieve used in molecular sieve centrifugal column described in step (2) is centrifuged column packing is 2 × 104~8 ×
106Da。
3. according to claim 2 quickly completely obtain mRNA ribosomes new polypeptide chain compound using molecular sieve centrifugal column
Method, it is characterised in that:
It is cycloheximide or chloramphenicol that the translation, which extends inhibitor,；
The concentration of the cycloheximide is 0.05~0.15mg/mL；
The concentration of the chloramphenicol is 0.05~0.15mg/mL；
The concentration of the Triton X-100 is percent by volume 0.1~1.5%.
4. according to claim 1 quickly completely obtain mRNA ribosomes new polypeptide chain compound using molecular sieve centrifugal column
Method, it is characterised in that:
When biological sample described in step (1) is eucaryote, the composition of the lysate is as follows: 15~25mM, pH7.4
HEPES-KOH solution, 10~20mM MgCl2, 150~250mM KCl, 50~150 μ g/ml cycloheximides, 1~3mM
DTT, 0.5~1.5% (v/v) Triton X-100；
When biological sample described in step (1) is prokaryotes, the composition of the lysate is as follows: 5~15mM, pH7.8
Tris-HCl, 5~15mM MgCl2, 30~70mM NH4Cl, 0.1~0.3% (v/v) TritonX-100,100 μ g/ml chlorine
Mycin.
5. according to claim 1 quickly completely obtain mRNA ribosomes new polypeptide chain compound using molecular sieve centrifugal column
Method, it is characterised in that:
Biological sample described in step (1) is prokaryotes, is also comprised the processing steps of: before the lysate is added
Lysozyme soln and MgCl are added in prokaryote2Solution processing.
6. according to claim 1 quickly completely obtain mRNA ribosomes new polypeptide chain compound using molecular sieve centrifugal column
Method, it is characterised in that:
Using the supernatant after molecular sieve centrifugation column purification cracking, specific step is as follows described in step (2):
(A) molecular sieve centrifugal column is taken out, column equilibration is carried out using column balance buffering liquid, is subsequently placed under 2~8 DEG C of environment and is filled out to column
Material sedimentation is complete；
(B) the molecular sieve centrifugal column settled, the solution being centrifuged off in column, the pillar prepared are taken out；
(C) supernatant after cracking is added drop-wise to molecular sieve centrifugal column column bed surface, filtrate is collected by centrifugation, obtains purified height
The mRNA ribosomes new polypeptide chain compound of purity.
7. according to claim 6 quickly completely obtain mRNA ribosomes new polypeptide chain compound using molecular sieve centrifugal column
Method, it is characterised in that:
Molecular sieve centrifugal column described in step (A) is MicroSpin S-400HR-GE Healthcare molecular sieve centrifugal column；
When biological sample described in step (A) is eucaryote, the composition of the column balance buffering liquid is as follows: 15~
HEPES-KOH solution, the 10~20mM MgCl of 25mM, pH7.42, 150~250mM KCl；
When biological sample described in step (A) is prokaryotes, the composition of the column balance buffering liquid is as follows: 5~15mM,
Tris-HCl, the 5~15mM MgCl of pH7.82, 30~70mM NH4Cl。
8. according to claim 6 quickly completely obtain mRNA ribosomes new polypeptide chain compound using molecular sieve centrifugal column
Method, it is characterised in that:
Sedimentation described in step (A) refers to 2~8 DEG C of gravitational settlings 6 hours or more completely；
The condition of centrifugation described in step (B) is to be centrifuged 1~3min in 500~700g；
The condition of centrifugation described in step (C) is to be centrifuged 30~90s in 500~700g.
9. according to any one of claims 1 to 8 quickly completely obtain mRNA ribosomes new polypeptide chain using molecular sieve centrifugal column
The method of compound builds the application in the sequencing of library in mRNA.
10. according to claim 9, using molecular sieve centrifugal column, quickly completely acquisition mRNA ribosomes new polypeptide chain is compound
The method of object builds the application in the sequencing of library in mRNA, it is characterised in that includes the following steps: that molecular sieve will be used by described
The mRNA ribosomes new polypeptide chain that the method that centrifugal column quickly completely obtains mRNA ribosomes new polypeptide chain compound obtains is compound
Object extracts, and obtains RNC-RNA, then carries out mRNA and builds library sequencing.
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| CN112111452A (en) * | 2020-07-05 | 2020-12-22 | 浙江大学 | Method for separating and purifying eukaryotic cell ribosome and using eukaryotic cell ribosome for in vitro translation |
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