CN106191296A - A kind of chromatin isolation technics of RNA purification - Google Patents
A kind of chromatin isolation technics of RNA purification Download PDFInfo
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- 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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The present invention relates to the chromatin isolation technics of a kind of RNA purification.Described method comprises the steps of and is first combined with magnetic bead by probe, then hybridizes with chromatin lysate, then repeatedly washs through gradient temperature.The present invention, by using BSA, random oligonucleotide primer to close magnetic bead in advance, reduces magnetic bead and albumen, the non-specific binding of RNA.In the present invention, first magnetic bead hatches with probe, hatches with albumen again, carry out gradient temperature washing after hybridization, it is ensured that the abundant bonding probes of magnetic bead combining target RNA, improve hybridization efficiency after removing excess probes.
Description
Technical field
The present invention relates to biological technical field, particularly relate to the chromatin isolation technics of a kind of RNA purification.
Background technology
Long-chain non-coding RNA (Long non-coding RNA, lncRNA) is transcribing more than 200 nucleotide
This, the overwhelming majority is positioned at nucleus.Although lncRNA does not encode any protein, but they at different tissues and grow rank
The expression of section still has specificity.LncRNA as a kind of new biomacromolecule in the generation evolution of human diseases
Play an important role.Therefore, the research molecular mechanism that plays a role of lncRNA just shows its necessity, good for disease
Diagnosis and treatment provide technical guarantee.
2011, Howard professor Chang of Stanford University developed the chromatin isolation technics of RNA purification
(chromatin isolation by RNA purification, ChIRP).ChIRP technology is a kind of in full-length genome scope
The method of the chromatin of interior qualification and RNA binding and protein interaction (Chu C, Qu K, Zhong FL, Artandi SE,
Chang HY.Genomic maps of long noncoding RNA occupancy reveal principles of
RNA-chromatin interactions.Mol Cell,2011,44(4):667–678.).Its principle is as follows: first with penta
Dialdehyde fixes cell, to maintain such as the rna regulations such as lncRNA and chromatinic interaction, then carries out cell cracking with ultrasonic
Broken, then hybridize with target lncRNA with biotin labeled oligonucleotide probe, mutual based on biotin and Streptavidin
Effect principle, with Streptavidin MagneSphere separate, purification chromatin complex, finally from the chromatin complex of purification
Separate protein, RNA or DNA, to carry out analysis (Chu C, Quinn J, the Chang HY.Chromatin in downstream
isolation by RNA purification(ChIRP).J Vis Exp,2012,(61):e3912.)。
ChIRP technology is widely used by the researchers of different field.ChIRP technology is utilized to pass through such as S Kan etc.
Research Hela cell successfully identifies lncRNA NEAT1 and acts on binding site (Kan S, the Taganov of genomic DNA
K,Rosenfeld J M,et al.Abstract 4881:Analysis of long-noncoding RNA
interaction at chromatin by chromatin isolation by RNA purification(ChIRP)
[J].Cancer Research,2015,75(15Supplement):4881-4881.).But, only in the level of domain
On carry out deeper into research, the function understanding lncRNA that could be more deep.2014, Chang etc. used ChIRP technology pin
Probe is designed in lncRNA particular result territory, have studied the roX1 needed for Male Drosophila dosage compensation effect, disclose roX1 merit
Can the composition structure in territory, i.e. three refer to palm structures, dissected this structure be responsible for function with chromatin interaction (Quinn J J,
Ilik I A,Qu K,et al.Domain ChIRP reveals the modularity of long noncoding RNA
architecture,chromatin interactions,and function[J].Nature Biotechnology,
2014,32(9):933-940.)。
There is a lot of shortcoming in existing ChIRP technology, as magnetic bead can adsorb nonspecific nucleic acid and protein, probe groups with
RNA hybridization inefficient etc., and then the specificity that result in ChIRP technology is low and sensitivity is the best.
Summary of the invention
In view of this, it is necessary to for above-mentioned problem, it is provided that the chromatin isolation technics of a kind of RNA purification.
To achieve these goals, the present invention adopts the following technical scheme that
The chromatin isolation technics (ChIRP technology) of a kind of RNA purification, comprises the steps of: first tied with magnetic bead by probe
Close, then hybridize with chromatin lysate, then repeatedly wash through gradient temperature.
Preferably, the chromatin isolation technics of RNA purification, comprise the steps of
S1, prepare chromatin lysate: crosslinking cell, cell lysis supersound process；
S2, magnetic bead pretreatment: include using BSA (bovine serum albumin) and random oligonucleotide primer to close strepto-affine
The magnetic bead of element labelling；
S3, prepare probe-bead complexes: be combined with biotin labeled DNA probe by the magnetic bead of pretreatment；
S4, ChIRP: joined by chromatin lysate in probe-bead complexes, hybridize 180min at 37 DEG C；Then
The each washing of 37 DEG C, 42 DEG C and 45 DEG C 2 times, wash 5min every time, obtain protein-probe-bead complexes；
S5, purification DNA: with the DNA Elution Buffer suspension protein-probe-bead complexes containing nuclease, incubate
Collect supernatant after educating, add E.C. 3.4.21.64, with phenol: chloroform: isoamyl alcohol extraction DNA, precipitate DNA with ethanol precipitation.
Preferably, in described step S1, the preparation method of chromatin lysate is:
S11, crosslinking cell
1. using the formaldehyde PBS re-suspended cell of 1v/v%, inhale and play mixing, room temperature shake rotates crosslinking 10 minutes, after being centrifuged again
Secondary crosslinking；
2. adding 1.25M glycine solution, room temperature shake rotates 5 minutes and terminates cross-linking reaction；It is centrifuged 5 minutes at 2000RCF,
Remove supernatant, with cold PBS Eddy diffusion cell, centrifugal segregation PBS；
S12, cell crack
1. with Swelling Buffer suspension cell, place 5 minutes on ice, lash mixing；
2. cell suspending liquid is centrifuged 5 minutes at 4 DEG C 2000～2500RCF, abandons supernatant；
3. with nucler lysis buffer re-suspended cell, place 10 minutes on ice；
S13, supersound process
By lysate in the supersonic frequency of 3s ON, 5s OFF ultrasonic 20 minutes, obtain chromatin lysate.
Preferably, the method for described step S2 magnetic bead pretreatment is: wash strepto-parent with 10mM Tris-HCl pH 7.5
With the magnetic bead 4 times of element labelling, wash 2 times with 1 × Hybridization Buffer afterwards, then add in magnetic bead containing BSA with
1 × TES of random oligonucleotide primer, removes TES solution after incubated at room 30min.
Preferably, in described TES, the concentration of BSA is 0.05-0.5wt%, and the concentration of random oligonucleotide primer is 10-20
μmol/L。
Preferably, the DNA sequence within random oligonucleotide primer is 20bp in described step S2.
Preferably, described step S3 is prepared the method for probe-bead complexes and is: by biotin-labelled DNA probe 85
After DEG C water places 3min, it is immediately placed on the DNA probe obtaining pretreatment on ice；It is added in the magnetic bead of pretreatment, and adds
Entering 2 × TES, 25 DEG C of gentle rotations mix 30-60min, remove supernatant；Wash magnetic bead twice with 1 × TES, remove supernatant, visited
Pin-bead complexes.
Preferably, ChIRP in described step S4 method particularly includes:
1. take 0.6mL chromatin lysate to be placed in EP pipe, add 1.2mL Hybridization Buffer, then add
Enter probe-bead complexes, 37 DEG C of hybridization 180min；
2. use the Wash Buffer containing 0.5mM PMSF each washing of 37 DEG C, 42 DEG C and 45 DEG C 2 times, wash every time
5min, obtains protein-probe-bead complexes.
Before carrying out ChIRP, chromatin lysate pre-washes, and after chromatin lysate room temperature is melted, adds 200 μ L
50% agarose beads, the vertical vortex mixer of room temperature condition processes 30～60min；Room temperature 4000～5000g is centrifuged 1min, in collection
Carry out clearly ChIRP.
Preferably, purification DNA in described step S5 method particularly includes:
1. with 150 μ L containing RNase A and RNase H DNA Elution Buffer suspension protein-probe-magnetic bead be combined
Thing, 37 DEG C of shakes are hatched 30 minutes, collect supernatant；
2. repeat step the most once, the supernatant of twice collection is merged；
3. adding 15 μ L E.C. 3.4.21.64 (proteinase K) mixings in supernatant, 55 DEG C of shakes hatch 45 minutes；
4. adding 300 μ L phenol: chloroform: isoamyl alcohol, be aggressively shaken 10 minutes, 16000RCF is centrifuged 5 minutes, phase of fetching water
Layer；
5. add 3 μ L nucleic acid settling agents, 30 μ L sodium acetates and 900 μ L 100% ethanol, mix-80 DEG C overnight；
6. it is centrifuged 30 minutes at 4 DEG C of 16100RCF, is carefully removed supernatant；Adding 1mL 70% ethanol, vortex mixes,
16100RCF is centrifuged 5 minutes, removes liquid, and drying at room temperature 1 minute is resuspended with 30 μ L deionized waters.
Preferably, in DNA Elution Buffer, the concentration that concentration is 0.1mg/mL, RNase H of RNase A is
0.1U/μL。
Preferably, every 0.75mL chromatin lysate needs to use the probe of 1 μ L 100pm/ μ L, the total probe of every 0.4nmol
Need to use 0.1mL magnetic bead.
Compared with prior art, there is advantages that
1. the present invention is by using BSA to close magnetic bead in advance, reduces the non-specific binding of magnetic bead and albumen；The present invention
By using random oligonucleotide primer to close magnetic bead in advance, reduce the non-specific binding of magnetic bead and DNA, enhance detection
Specificity.The size of BSA and random oligonucleotide primer is just right, can close the specificity of magnetic bead Enhancement test, again will not
Excessive and affect the combination of probe and albumen.
2. in the present invention, first magnetic bead hatches with probe, hatches with albumen again, carry out after hybridization after removing excess probes
Gradient temperature washs, it is ensured that the abundant bonding probes of magnetic bead combining target RNA, improves hybridization efficiency.Additionally, relatively conventional probe
First hatching with albumen and hatch with magnetic bead or method that probe, albumen and pearl are hatched simultaneously again, the present invention has saved magnetic greatly
The usage amount of pearl, saves experimental cost.
Accompanying drawing explanation
Fig. 1 is prior art ChIRP technical schematic diagram.
Fig. 2 is ChIRP technical schematic diagram of the present invention.
Fig. 3 is that in embodiment 1, ChIRP technology separates chromatinic electrophoretogram.
Wherein, figure a is for using ChIRP technology for detection TGFBI genetic results figure of the present invention, and figure b is for using ChIRP of the present invention
Technology for detection GAPDH genetic results figure, figure c is for using existing ChIRP technology for detection TGFBI genetic results figure.Every width figure is swum
Road 1 is Maker, and swimming lane 2 is 10%Input group, and swimming lane 3 is ChIRP group, and swimming lane 4 is negative control group (NC group).
Fig. 4 is that in embodiment 2, ChIRP technology separates chromatinic electrophoretogram.
Wherein, figure a is for using ChIRP technology for detection NIPA1 genetic results figure of the present invention, and figure b is for using ChIRP of the present invention
Technology for detection GAPDH genetic results figure, figure C is for using existing ChIRP technology for detection NIPA1 genetic results figure.Every width figure is swum
Road 1 is Maker, and swimming lane 2 is 10%Input group, and swimming lane 3 is ChIRP group, and swimming lane 4 is negative control group (NC group).
Detailed description of the invention
In order to better illustrate the present invention, it is described further with detailed description of the invention below in conjunction with the accompanying drawings.In the present invention
Agents useful for same or instrument all can be buied by market, and the detection method of use such as PCR, agarose gel electrophoresis etc. are all this area institutes
Known to, do not repeat them here.
Embodiment 1
The present embodiment carries out ChIRP experiment for TINCR.TINCR is long-chain non-coding RNA, and species are the mankind, target gene
For TGFBI, experiment material is human epidermal tissue.
The chromatin isolation technics of a kind of RNA purification, comprises the following steps:
S1, prepare chromatin lysate
S11, crosslinking cell
The institute of crosslinking cell the most at room temperature performs.The formaldehyde PBS of 1v/v% by 37% formaldehyde and PBS according to 27:
The volume ratio configuration of 973 forms, must be now with the current.
1. with the formaldehyde PBS suspension cell of 1v/v%, inhaling and play mixing, room temperature shake rotation cross-links 10 minutes, 1500～
2000rpm cross-links after being centrifuged 5min again；
2. terminating cross-linking reaction, glycine (about 0.01g) the solution room temperature shake adding 0.1mL1.25M rotates 5 minutes；?
2000RCF is centrifuged 5 minutes, removes supernatant, with cold PBS Eddy diffusion cell, then is centrifuged 5 minutes at 2000RCF；Be repeated once from
The heart, removes PBS as far as possible, keeps in or directly carry out further experiment for-80 DEG C.
S12, cell crack
1. with cell after 1mL Swelling Buffer suspension crosslinking, put 5 minutes on ice, take out with No. 4.5 syringe needles of syringe
Play 15-20 mixing；If cell is cold preservation sample after Jiao Lian, need to first thaw, centrifugal 5 minutes of 2500RCF 4 DEG C, it is eliminated as much as
Remaining PBS, adds Swelling Buffer afterwards and carries out corresponding operating；
2. cell suspending liquid is centrifuged 5 minutes at 4 DEG C of 2000-2500RCF；
3. put 10 minutes on ice with the nucler lysis buffer re-suspended cell of 2mL, with further cell lysis.
S13, supersound process
1. taking out 20 μ L lysates, remaining lysate forwards in ultraphonic pipe, condition of ice bath 3s ON, 5s OFF ultrasonic 20
Minute, obtain chromatin lysate ,-80 DEG C of storages；
2. take the 10～20 ultrasonic samples of μ L and carry out 1% agarose gel electrophoresis, deposition condition 100v electricity with the most ultrasonic sample
Swimming 20～35min.After electrophoresis checking, band is at about 300bp, it was demonstrated that ultrasonic respond well.
S2, magnetic bead pretreatment
1. take appropriate magnetic bead to be placed in clean EP pipe, be placed on magnetic frame and remove storage liquid；
2. suspend magnetic bead with 1mL 10mM Tris-HCl pH 7.5, remove supernatant at magnetic frame；
3. repeat 2. to wash 4 times；
4. wash 2 times with 1mL 1 × Hybridization Buffer, remove supernatant at magnetic frame；
5. to magnetic bead add 1ml containing 0.05-0.5wt%BSA and 10-20 μm ol/L random oligonucleotide primer 1 ×
TES, incubated at room 30min is placed on magnetic frame removal TES solution.Within described random oligonucleotide primer is 20bp
DNA sequence.
S3, prepare probe-bead complexes
S31, probe pretreatment
The present embodiment designs a plurality of probe composition probe groups for lncRNA, the Probe components of experimental group be Even group with
Each 5 probes of Odd group, Even group and Odd group, blank group (NC group) contains 5 probes (being shown in Table 1).Before pretreatment, will
The probe groups of experimental group and blank group is configured to the probe solution that whole concentration and probe concentration is 100pm/ μ L.
Appropriate probe solution need to be taken put into 85 by the ratio of 1 μ L 100pm/ μ L probe solution according to every 0.75mL chromatin
DEG C water in degeneration 3 minutes, be immediately placed in afterwards on ice, obtain the probe of pretreatment.
Table 1, embodiment 1 middle probe form
S32, probe are combined with magnetic bead
1. the ratio of the magnetic bead of 0.1mL is needed according to total probe of every 0.4nmol, by probe (experimental group and the moon of pretreatment
Property matched group) add in the magnetic bead of pretreatment, and add 100 μ L 2 × TES, 25 DEG C rotate gentleness mixing 30min, at magnetic frame
Upper standing 1min, collects magnetic bead, abandons supernatant；
2. adding 0.35mL 1 × TES in magnetic bead, 25 DEG C of gentle rotations are washed 5min, are stood 1min on magnetic frame,
Collect magnetic bead, abandon supernatant；
3. repeat step the most once, obtain probe-bead complexes (experimental group and negative control group).
S4、ChIRP
S41, ChIRP pre-wash
1., after-80 DEG C of chromatin lysate room temperatures preserved are melted, 200 μ L 50% agarose beads, room temperature condition are added
Vertical vortex mixer processes 30～60min；
2. room temperature 4000～5000g is centrifuged 1min, collects supernatant for pre-washing sample；
S42, ChIRP molecule hybridizes
1. take 0.6mL to pre-wash sample and be placed in 2mL EP pipe, add 1.2mL Hybridization Buffer；?
Probe-the magnetic bead of experimental group and negative control group is added separately in corresponding EP pipe, 37 DEG C of hybridization incubation in molecule hybrid heater
180min；
2. use the Wash Buffer containing 0.5mM PMSF to carry out gradient temperature repeatedly to wash: 37 DEG C are washed 2 times, 42 DEG C
Wash and wash for 2 times, 45 DEG C 2 times, collect magnetic bead with standing 1min on vertical vortex mixer shake washing 5min magnetic frame every time and abandon
Supernatant (Wash Buffer is used in washing, is preheating to corresponding wash temperature before use, and adds PMSF).
S5, purification DNA
Prepare this experiment DNA Elution Buffer before experiment, add by every 150 μ L DNA Elution Buffer
The ratio entering 1.5 μ L RNase A (10mg/mL) and 1.5 μ L RNase H (10U/ μ L) adds RNase A and RNase H, whirlpool
Rotation mixing, the final concentration of 0.1U/ μ L of final concentration of 0.1mg/mL, the RNase H of RNase A.RNase A and RNase H fall
Solve RNA, DNA-protein complex is separated with probe-bead complexes.
1. with 150 μ L above-mentioned DNA Elution Buffer suspension protein-probe-bead complexes, 30 points are hatched for 37 DEG C
Clock, midfeather 2～5min is shaken once；Stand 1min on magnetic frame and combine magnetic bead, collect supernatant；
2. repeat step 2., merge twice collection supernatant (about 300 μ L, compound for DNA-protein in supernatant
Thing)；
The most each sample adds 15 μ L 20mg/ml E.C. 3.4.21.64 (proteinase K, PK) mixings, and 55 DEG C of shakes hatch 45 points
Clock, with the protein in supernatant of degrading；
The most each sample adds 300 μ L phenol: chloroform: isoamyl alcohol (PhOH:Chloroform:Isoamyl), is aggressively shaken 10
Minute, room temperature 16000RCF is centrifuged 5 minutes and takes upper strata aqueous phase (about 300 μ L)；
5. 3 μ L nucleic acid settling agent (Acryl Carrier, AC), 30 μ L sodium acetate (NaOAc), and 900 μ L 100% second are added
Alcohol (EtOH), mixes-80 DEG C overnight；
6. it is centrifuged 30 minutes at 4 DEG C of 16100RCF, is carefully removed supernatant；Adding 1mL 70% ethanol, vortex mixes,
16100RCF is centrifuged 5 minutes, removes liquid as far as possible, and drying at room temperature 1 minute is resuspended with 30 μ L deionized waters.
The DNA obtaining purification carries out PCR, uses agggctagcagtgccaagta and ctgcaacagtacctgccaga
Respectively as upstream and downstream primer, carry out agarose gel electrophoresis detection afterwards.100 μ L are used to pre-wash sample as input group.
In order to better illustrate beneficial effects of the present invention, inventor has also carried out two example contrast tests.Comparative example 1 uses the present invention
ChIRP technology, but detection gene is GAPDH (compareing as gene negative).Comparative example 2 uses ChIRP technology in prior art
(Chu C,Quinn J,Chang H Y.Chromatin isolation by RNA purification(ChIRP)[J]
.Journal of Visualized Experiments, 2012,61 (61): 3912.) to test, detection gene is
TGFBI.Result is as shown in Figure 2.
The preparation method of chromatin lysate in comparative example 2, probe pretreatment method identical with the present invention.But magnetic bead
Do not carry out BSA and random oligonucleotide primer is closed.0.6mL chromatin lysate will add 1.2mL Hybridization
In Buffer, adding correspondent probe, 65 DEG C of incubation degeneration 10min, 37 DEG C of hybridization 180min, add latter 25 DEG C of magnetic bead mixing and incubate
Educate 60min, wash magnetic beads 5 times with the Wash Buffer containing 0.5mM PMSF at 25 DEG C, hatch 5min, use magnetic force for each 25 DEG C
Frame removes supernatant.The purification process of DNA sample is identical with the present invention.
From the figure 3, it may be seen that the band of the ChIRP group of the present embodiment is clear, without conditions of streaking, band concentration is substantially and Input
Group is quite；The ChIRP group of comparative example 1 is without band；ChIRP group band in comparative example 2 is unintelligible, has conditions of streaking.Therefore, originally
Invention ChIRP technology has higher specificity and sensitivity.
Embodiment 2
The present embodiment carries out ChIRP experiment for FENDRR.FENDRR is long-chain non-coding RNA, and species are the mankind, target base
Because NIPA1, experiment material is hLF cell.
The chromatin isolation technics of a kind of RNA purification, comprises the following steps:
S1, prepare chromatin lysate
Identical with step described in embodiment 1.
S2, magnetic bead pretreatment
Identical with step described in embodiment 1.
S3, prepare probe-bead complexes
S31, probe pretreatment
The present embodiment designs a plurality of probe composition probe groups for lncRNA, the Probe components of experimental group be Even group with
Each 5 probes of Odd group, Even group and Odd group, blank group (NC group) contains 5 probes (being shown in Table 1).Before pretreatment, will
The probe groups of experimental group and blank group is configured to the probe solution that middle probe concentration is 100pm/ μ L.
Appropriate probe solution need to be taken put into 85 by the ratio of 1 μ L 100pm/ μ L probe solution according to every 0.75mL chromatin
DEG C water in degeneration 3 minutes, be immediately placed in afterwards on ice, obtain the probe of pretreatment.
Table 1, embodiment 1 middle probe form
S32, probe are combined with magnetic bead
1. need the ratio of the magnetic bead of 0.1mL according to total probe of every 0.4nmol, the probe of pretreatment is added pretreatment
In magnetic bead, and adding 100 μ L2 × TES, 25 DEG C rotate gentleness mixing 30min；
2. collecting magnetic bead on magnetic frame and remove supernatant, adding 0.35mL 1 × TES, gentle rotation is washed magnetic bead, is placed in
Cleaning mixture is removed on magnetic frame；
3. repeat step the most once, obtain probe-bead complexes.
S4、ChIRP
Identical with step described in embodiment 1.
S5, purification DNA
Identical with step described in embodiment 1.
The DNA obtaining purification carries out PCR, uses ttggaggtaaaagccaccac and gctgccaggttttgttgtct
Respectively as upstream and downstream primer, carry out agarose gel electrophoresis detection afterwards.100 μ L are used to pre-wash sample as input group.
In order to better illustrate beneficial effects of the present invention, inventor has also carried out two example contrast tests.Comparative example 1 uses the present invention
ChIRP technology, but detection gene is GAPDH；Comparative example 2 use ChIRP technology in prior art (Chu C, Quinn J,
Chang H Y.Chromatin isolation by RNA purification(ChIRP)[J].Journal of
Visualized Experiments, 2012,61 (61): 3912.) to test, detection gene is NIPA1.Result such as Fig. 4 institute
Show.
From the figure 3, it may be seen that the band of the ChIRP group of the present embodiment is clear, without conditions of streaking, band concentration is substantially and Input
Group is quite；The ChIRP group of comparative example 1 is without band；ChIRP group band in comparative example 2 is unintelligible, has conditions of streaking.Therefore, originally
Invention ChIRP technology has higher specificity and sensitivity.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed, but also
Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that, for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement, these broadly fall into the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. the chromatin isolation technics of a RNA purification, it is characterised in that comprise the steps of and probe is first combined with magnetic bead,
Hybridize with chromatin lysate again, then repeatedly wash through gradient temperature.
The chromatin isolation technics of RNA purification the most according to claim 1, it is characterised in that comprise the steps of
S1, prepare chromatin lysate: crosslinking cell, cell lysis supersound process；
S2, magnetic bead pretreatment: include the magnetic bead using BSA and random oligonucleotide primer to close marked by streptavidin；
S3, prepare probe-bead complexes: be combined with biotin labeled DNA probe by the magnetic bead of pretreatment；
S4, ChIRP: joined by chromatin lysate in probe-bead complexes, hybridize 180min at 37 DEG C；Then 37
DEG C, 42 DEG C and 45 DEG C of each washings 2 times, wash 5min every time, obtain protein-probe-bead complexes；
S5, purification DNA: with the DNA Elution Buffer suspension protein-probe-bead complexes containing nuclease, after hatching
Collect supernatant, add E.C. 3.4.21.64, with phenol: chloroform: isoamyl alcohol extraction DNA, precipitate DNA with ethanol precipitation.
The chromatin isolation technics of RNA purification the most according to claim 2, it is characterised in that dye in described step S1
The preparation method of matter lysate is:
S11, crosslinking cell
1. using the formaldehyde PBS re-suspended cell of 1v/v%, inhale and play mixing, room temperature shake rotates crosslinking 10 minutes, again hands over after being centrifuged
Connection；
2. adding 1.25M glycine solution, room temperature shake rotates 5 minutes and terminates cross-linking reaction；It is centrifuged 5 minutes at 2000RCF, goes
Clearly, with cold PBS Eddy diffusion cell, centrifugal segregation PBS；
S12, cell crack
1. with Swelling Buffer suspension cell, place 5 minutes on ice, lash mixing；
2. cell suspending liquid is centrifuged 5 minutes at 4 DEG C of 2500RCF, abandons supernatant；
3. with nucler lysis buffer re-suspended cell, place 10 minutes on ice；
S13, supersound process
By lysate in the supersonic frequency of 3s ON, 5s OFF ultrasonic 20 minutes, obtain chromatin lysate.
The chromatin isolation technics of RNA purification the most according to claim 2, it is characterised in that described step S2 magnetic bead is pre-
The method processed is: wash the magnetic bead 4 times of marked by streptavidin with 10mM Tris-HCl pH 7.5, afterwards with 1 ×
Hybridization Buffer washs 2 times, then adds containing BSA and 1 × TES of random oligonucleotide primer, room in magnetic bead
Temperature removes TES solution after hatching 30min.
The chromatin isolation technics of RNA purification the most according to claim 4, it is characterised in that in described TES, BSA's is dense
Degree is 0.05-0.5wt%, and the concentration of random oligonucleotide primer is 10-20 μm ol/L.
The chromatin isolation technics of RNA purification the most according to claim 4, it is characterised in that few core in described step S2
Thuja acid random primer is the DNA sequence within 20bp.
The chromatin isolation technics of RNA purification the most according to claim 2, it is characterised in that the preparation of described step S3 is visited
The method of pin-bead complexes is: after biotin-labelled DNA probe is placed 3min in 85 DEG C of water, is immediately placed on and obtains on ice
DNA probe to pretreatment；Being added in the magnetic bead of pretreatment, and add 2 × TES, 25 DEG C of gentle rotations mix 30-
60min, removes supernatant；Wash magnetic bead twice with 1 × TES, remove supernatant, obtain probe-bead complexes.
The chromatin isolation technics of RNA purification the most according to claim 2, it is characterised in that ChIRP in described step S4
Method particularly includes:
1. take 0.6mL chromatin lysate to be placed in EP pipe, add 1.2mL Hybridization Buffer, add spy
Pin-bead complexes, 37 DEG C of hybridization 180min；
2. use the Wash Buffer containing 0.5mM PMSF each washing of 37 DEG C, 42 DEG C and 45 DEG C 2 times, wash 5min every time,
To protein-probe-bead complexes.
The chromatin isolation technics of RNA purification the most according to claim 8, it is characterised in that before carrying out ChIRP, dye
Chromaticness lysate pre-washes, and after chromatin lysate room temperature is melted, adds 200 μ L50% agarose beads, room temperature condition
Vertical vortex mixer processes 30～60min；Room temperature 4000～5000g is centrifuged 1min, collects supernatant and carries out ChIRP.
The chromatin isolation technics of RNA purification the most according to claim 2, it is characterised in that purification in described step S5
DNA's method particularly includes:
1. the DNA Elution Buffer suspension protein-probe-bead complexes of RNase A and RNase H is contained with 150 μ L,
37 DEG C of shakes are hatched 30 minutes, collect supernatant；
2. repeat step the most once, the supernatant of twice collection is merged；
3. adding 15 μ L E.C. 3.4.21.64 mixings in supernatant, 55 DEG C of shakes hatch 45 minutes；
4. adding 300 μ L phenol: chloroform: isoamyl alcohol, be aggressively shaken 10 minutes, 16000RCF is centrifuged 5 minutes, aqueous layer；
5. add 3 μ L nucleic acid settling agents, 30 μ L sodium acetates and 900 μ L 100% ethanol, mix-80 DEG C overnight；
6. it is centrifuged 30 minutes at 4 DEG C of 16100RCF, is carefully removed supernatant；Adding 1mL 70% ethanol, vortex mixes, 16100RCF
Centrifugal 5 minutes, remove liquid, drying at room temperature 1 minute, resuspended with 30 μ L deionized waters.
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