CN106168625A - A kind of RNA pulldown method and test kit - Google Patents
A kind of RNA pulldown method and test kit Download PDFInfo
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- CN106168625A CN106168625A CN201610635149.6A CN201610635149A CN106168625A CN 106168625 A CN106168625 A CN 106168625A CN 201610635149 A CN201610635149 A CN 201610635149A CN 106168625 A CN106168625 A CN 106168625A
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Abstract
The present invention relates to a kind of RNA pulldown method and test kit.Described method comprises the steps of S1, magnetic bead pretreatment: use BSA and random oligonucleotide primer to close the magnetic bead containing Streptavidin；S2, prepare probe bead complexes；S3, extraction pretreatment cell whole protein: hatch to extracting addition ribonuclease in cell whole protein sample, add magnetic bead and hatch, collect supernatant；S4、pulldown；The collection of S5, RNP.There is advantages that the present invention to pass through in advance and use BSA, random oligonucleotide primer to close magnetic bead, reduce magnetic bead and albumen, the non-specific binding of RNA.The present invention is protected by systematicness nuclease, prevents the pollution of nuclease, adds stability and the reliability of experiment, reproducible, simplifies experiment flow.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of RNA for studying RNA and protein-interacting
Pulldown method and test kit.
Background technology
Rna binding protein (RBPs) plays an important role in the expression of post-transcriptional level regulator gene, concrete effect
Including montage, the polyadenylated and maintenance of stability of mRNA precursor, mRNA is from nucleus to cytoplasmic transport, and mRNA determines
Position and translation etc..RBPs regulation effect during these is the spy by combining the transcript after newly synthesized or maturation
Fixed sequence realizes.Simultaneously, there is the specific nucleotide sequence that a lot of RBPs is capable of identify that in RNA.In order to study RNA and egg
White interaction establishes a lot of method, and these methods include that EMSA, SELEX technology, RNA footprinting, RNA exempt from
Epidemic disease precipitation (RIP) and RNA pulldown technology.
RNA pulldown technology utilizes de-end-labelled RNA efficiently concentrating and identifies rna binding protein (RBPs).This
Method avoids the use of antibody, significantly extends range.The most perfect along with RNA pulldown technology, it is being given birth to
In life scientific research and medical domain, role is the most important.
Hirofumi etc. isolate mRNA by RNA pulldown technology from the minimal amount of sensory neuron of nematicide,
The gene expression profile of sensory neuron has been identified subsequently by chip analysis.(KUNITOMO H,UESUGI H,KOHARA Y,
et al.2005.Identification of ciliated sensory neuron-expressed genes in
Caenorhabditis elegans using targeted pull-down of poly(A)tails.Genome Biol
[J],6:R17.)
In present research, the probe of RNA pulldown research is mainly the single-stranded probe of synthesizing biotinylated labelling,
Expand the research range of RNA.Tsai etc. pass through the RNA pulldown technical Analysis of improvement long-chain non-coding RNA
(lincRNA) regulation chromatin state and epigenetic.They find that lincRNA HOTAIR repaiies as at least two histone
The scaffold of decorations complex.5 ' the ends of HOTAIR combine many combs suppression complex 2 (PRC2), and 3 ' ends combine LSD1/CoREST/
REST complex, completes the assembling of RPC2 and the LSD1 complex of RNA mediation thus completes two sites of H3K27 and H3K4
Methylate.(TSAI M C,MANOR O,WAN Y,et al.2010.Long noncoding RNA as modular
scaffold of histone modification complexes.Science[J],329:689-693.)
Above-mentioned RNA pulldown method has an obvious shortcoming: the pearl (agarose beads used in (1) experiment
Or magnetic bead) and the non-specific binding of albumen, easily cause the false positive of experimental result；(2) rna probe is special with protein bound
Property is the highest；(3) experimentation also suffers from the impact that nuclease pollutes.
Summary of the invention
In view of this, it is necessary to for above-mentioned problem, it is provided that a kind of RNA pulldown method and test kit.
To achieve these goals, the present invention adopts the following technical scheme that
A kind of RNA pulldown method, comprises the steps of
S1, magnetic bead pretreatment: use BSA (bovine serum albumin) and random oligonucleotide primer to close Streptavidin mark
The magnetic bead of note；
S2, prepare probe-bead complexes: be combined with biotin labeled rna probe by the magnetic bead of step S1 pretreatment；
S3, extraction pretreatment cell whole protein: in the cell whole protein extracted, add deoxyribonuclease hatch,
Add unpretreated magnetic bead to hatch, collect supernatant；
S4, pulldown: the supernatant that step S3 is collected is joined in probe-bead complexes prepared by step S2, adds
Enter and hatch containing deoxyribonuclease inhibitor and ribonuclease inhibitor, collect magnetic bead, use qiagen rnase enzyme inhibitor
NT2buffer wash magnetic bead, obtain protein-probe-bead complexes；
The collection of S5, RNP (protein-probe complex): protein-probe-bead complexes step S4 prepared adds
Urea CHAPS buffer is hatched, and collects supernatant and i.e. obtains RNP.
Preferably, the method for described step S1 magnetic bead pretreatment is: wash 80 μ L Streptavidin marks with 1mL 1 × TES
The magnetic bead of note, removes TES cleaning mixture, then adds 1mL in magnetic bead containing BSA and 1 × TES of random oligonucleotide primer, room temperature
TES solution is removed after hatching 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 S1.
Preferably, described step S2 is prepared the method for probe-bead complexes and is: will contain 2-5 μ g biotin labeling RNA and visit
The solution of pin places 2min at 90 DEG C, is immediately placed on 2min on ice, adds 50 μ L RNA structure buffer and 20 μ L
DEPC water, room temperature is placed 20min and is prepared the rna probe of secondary structure；It is added in the magnetic bead of pretreatment, and adds
100 μ L 2 × TES, 25 DEG C of gentle rotations mix 30-60min, remove supernatant；Wash magnetic bead twice with 1 × TES, remove TES.
Preferably, in described step S3, the method for pretreatment cell whole protein is: to 1.5 × 107Individual cell extraction obtains
Cell whole protein extract in add 10 μ L DNase and 3.2 μ L DNase Buffer, room temperature gentleness hatches 1h；Add
The 40 unpretreated magnetic beads of μ L, 4 DEG C of gentle rotation 30min；Collect the cell whole protein that supernatant is pretreatment.
Preferably, the method for described step S3 extraction cell whole protein is: wash 1.5 × 10 with PBS7Individual cell is once；
Add 975 μ L RIP Buffer, 10 μ L PMSF solution, 10 μ L protease inhibitor cocktail and 5 μ L DTT
Solution, homogenizer is homogenized 15-20 time；4 DEG C, 1500g be centrifuged 15min, collect supernatant and be cell whole protein extract.
Preferably, described step S4pulldown method particularly includes: cell whole protein step S3 prepared joins
In probe-bead complexes, and add 5 μ L RNase inhibitor, 15 μ g poly (dI dC), 5 μ L0.5M EDTA (second
Ethylenediamine tetraacetic acid (EDTA)) solution and 2.5 μ L 0.5M EGTA (ethylene glycol diethyl ether ethylenediamine tetraacetic acid (EDTA)) solution；Room temperature gentleness rotates hatches
2h, collects magnetic bead；Add 974 μ L NT2buffer, 10 μ L PMSF solution, 10 μ L protease inhibitor
Cocktail, 5 μ L DTT solution and 1 μ L RNase inhibitor, wash magnetic bead, repeats washing four times.The present invention selects
With EDTA as the inhibitor of deoxyribonuclease.
Preferably, in described step S5, the collection method of RNP is: add 40 μ L in protein-probe-bead complexes
Urea CHAPS buffer, hatches 5-10h for 4 DEG C, collects supernatant and is probe-albumen composition.
Preferably, the concentration of described DTT solution is 100mM, and the concentration of described PMSF solution is 10mg/mL.
A kind of RNA pulldown test kit, comprises following reagent:
Magnetic bead and reagent treatment thereof: 2 × TES solution, the magnetic bead of marked by streptavidin, random oligonucleotide primer；Institute
The concentration stating random oligonucleotide primer is 10-20 μm ol/L；
RNA reagent treatment: DNase, DNase Buffer, RNase inhibitor；
Protein extraction and reagent treatment: RIP buffer, Urea CHAPS buffer, protease inhibitor
Cocktail, DTT solution, PMSF solution；The concentration of described DTT solution is 100mM, and the concentration of described PMSF solution is 10mg/
mL；
Probe and combine reagent treatment: RNA structure buffer, NT2buffer, poly (dI dC).
The present invention is directed to target area design specific RNA probe, probe is through desthiobiotin labelling；Streptavidin
It is coupled on magnetic bead, and and the affine combination of desthiobiotin；Cell whole protein is hatched with magnetic bead-probe complex, action protein
Molecule can be specific binding with rna probe；Non-specific binding protein molecular can be removed through washing；Finally, washing
De-liquid (for Streptavidin) carries out eluting, obtains purpose probe-albumen composition, then through Western Blot or mass spectrum
Identify protein types.
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 geneome RNA.BSA
Just right with the size of random oligonucleotide primer, can close the specificity of magnetic bead Enhancement test, again will not be excessive and affect
Probe and the combination of albumen.
2. the present invention uses deoxyribonuclease pretreatment protein sample, uses unpretreated magnetic bead to protein sample
Pre-wash, remove the internal DNA in protein sample, prevent it to be wound around magnetic bead thus affect the RNA being coupled on magnetic bead and visit
Pin and the combination of protein, then use deoxyribonuclease inhibitor to inactivate deoxyribonuclease.The present invention passes through system
Property nuclease protection, prevent the pollution of nuclease, add stability and the reliability of experiment, reproducible, simplify experiment
Flow process.
3. in the present invention, first magnetic bead hatches with probe, hatches with albumen after removing excess probes again, and relatively conventional probe is first
Hatch with albumen and hatch with magnetic bead again or probe, albumen and pearl hatch the usage amount having saved magnetic bead greatly simultaneously, save
Experimental cost.The present invention uses magnetic bead to instead of sepharose 4B, saves centrifugal step, saves test period.Meanwhile, magnetic is used
The washing of pearl method instead of the step that sepharose 4B is centrifugal, and can make that supernatant removes is more thorough, improves the specificity reacted.
Accompanying drawing explanation
Fig. 1 is distinct methods pulldown design sketch.
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 etc. is all known in the art, do not repeat them here.
Embodiment 1
As a example by the lincRNA HOTAIR probe pulldown effect to LSD1 albumen, the present invention is described, probe warp
Cross desthiobiotin 8-oxoG labelling.The design of probe and labeling method are well-known to those skilled in the art, at this no longer
Repeat.Streptavidin is coupled at magnetic bead (BeaverBeadsTMStreptavidin, castor bio tech ltd) on, and
Combination affine with desthiobiotin；Cell whole protein is hatched with magnetic bead-rna probe, and action protein molecule can be special with probe
Property combine；Non-specific binding protein molecular can be removed through washing；Finally, then with eluent (for Streptavidin)
Carry out eluting, obtain purpose probe-albumen composition, then identify protein types through Western Blot.Concrete steps are such as
Under:
S1, magnetic bead pretreatment
1. take the 80 μ L magnetic bead containing Streptavidin to be placed in without in RNase EP pipe, add 1mL 1 × TES and wash magnetic bead；
2. it is placed on magnetic frame removal TES cleaning mixture, adds 1mL containing 0.5%BSA and 10 μm ol/L oligonucleotide to magnetic bead
1 × TES of random primer, incubated at room 30min is placed on magnetic frame removal TES solution.Described random oligonucleotide primer
For the DNA sequence within 20bp.
S2, prepare probe-bead complexes
1. by the DNA probe of 2-5 μ g size 300-1000bp with 30 μ L DEPC water dissolutioies, 2min is placed at 90 DEG C, immediately
Being placed in 2min on ice, add 50 μ LRNA structure buffer and 20 μ L DEPC water, room temperature is placed 20min and is prepared
The rna probe of secondary structure；
2. being added by the rna probe of secondary structure in the magnetic bead of pretreatment, and add 100 μ L 2 × TES, 25 DEG C rotate temperature
With mixing 30min, it is placed in magnetic frame and gets on supernatant；
3. adding 0.35mL 1 × TES, 25 DEG C of gentle rotations are washed magnetic bead 5min, are placed on magnetic frame removal cleaning mixture；
4. step is repeated the most once.
S3, extraction pretreatment cell whole protein
S31, extraction cell whole protein
1. 1.5 × 10 are washed with PBS7Individual Hela cell is once；
Add 975 μ L RIP Buffer, 10 μ L PMSF solution, 10 μ L protease inhibitor cocktail and
5 μ L DTT solution, homogenizer is homogenized 15-20 time；4 DEG C, 1500g be centrifuged 15min, collect supernatant and be cell whole protein and extract
Thing；The concentration of described DTT solution is 100mM, and the concentration of described PMSF solution is 10mg/mL；.
S32, pretreatment cell whole protein
1. in the cell whole protein extract of above-mentioned preparation, add 10 μ L DNase, 3.2 μ L DNase Buffer, room
25 DEG C of gentlenesses of temperature hatch 1h；
2. in protein sample, add the 40 unpretreated magnetic beads of μ L, 4 DEG C of gentle rotation 30min；
3. collect magnetic bead on magnetic frame, the cell whole protein of supernatant pretreatment is transferred in new EP pipe.
S4、pulldown
1. the cell whole protein of preparation is joined in probe-bead complexes, and add 472.5 μ L NT2buffer, 5
μ L RNase inhibitor, 15 μ g poly (dI dC), 5 μ L 0.5M EDTA and 2.5 μ L 0.5M EGTA；
2. room temperature gentleness rotates and hatches 2h；
3. collect magnetic bead on magnetic frame, remove supernatant；
4. add 974 μ L NT2buffer, 10 μ L PMSF solution, 10 μ L protease inhibitor cocktail,
5 μ L DTT solution and 1 μ L RNase inhibitor, wash magnetic bead, removes the albumen of non-specific binding, repeats washing four
Secondary, obtain protein-probe-bead complexes；The concentration of described DTT solution is 100mM, and the concentration of described PMSF solution is
10mg/mL；.
S5, RNP collect
1. in protein-probe-bead complexes, add 40 μ L Urea CHAPS buffer, hatch 5-10h for 4 DEG C.
2. collecting magnetic bead on magnetic frame, supernatant is probe-albumen composition, is transferred in new EP pipe by supernatant RNP.
3. product is placed in-80 DEG C of preservations, analyzes for further Western Blot.
In order to better illustrate the effect of the present invention, inventor has also carried out two groups of contrast experiments.
Comparative example 1 uses PierceTMMagnetic RNA-Protein Pull-Down Kit method, referring specifically to this examination
Agent box description.
Comparative example 2 uses method (the TSAI M C, MANOR O, WAN Y, et al. 2010.Long that Tsai et al. reports
noncoding RNA as modular scaffold of histone modification complexes.Science
[J], 329:689-693.), concretely comprise the following steps:
(1) biotin labeled RNA mixture (Roche) in vitro transcription is utilized to obtain by t7 rna polymerase (Promega)
Obtaining biotin labeled RNAs, by the DNase I (Promega) without RNase, RNeasy Mini Kit (QIAGEN) is right
RNA product carries out isolated and purified.Take 3 μ g biotinylated rnas, 90 DEG C of heating 2min, be placed on 2min on ice, change RNA structure into and delay
Rush liquid (10mM Tris pH 7,0.1M KCl, 10mM MgCl2), be subsequently placed in room temperature 20min and form it into correct two grade
Structure.
(2)107Individual HeLa cell is resuspended by 2mL PBS, adds 2mL nuclear isolation buffer (1.28M
sucrose；40mM Tris-HCl pH 7.5；20mM MgCl2；4%Triton X-100) and the water of 6mL be placed on ice
20min, period often mixes.Nucleus can be centrifuged 15min by 2,500g and obtain.Nucleus precipitation is resuspended in 1mLRIP and delays
Rush liquid (150mM KCl, 25mM Tris pH 7.4,0.5mM DTT, 0.5%NP40,1mM PMSF and protease
In Inhibitor (Roche Complete Protease InhibitorCocktail Tablets).Resuspended nucleus leads to
Crossing Potter-Elvehjem Tissue Grinders and carry out 15-20 mechanical homogenisation, nuclear membrane and fragment by 13,000 turn, centrifugal 10min separates.
(3) RNA and the 1mg Hela nucleus extraction thing folded is mixed in RIP, hatch 1h at room temperature, take washed
Biotinylated agarose beads (Invitrogen) add in the system of each association reaction, be further incubated at room temperature
1h.Pearl is washed 5 times in Handee spin pillar, adds SDS buffer and boils and Western Blot detection.
The method using the embodiment of the present invention and comparative example separately verifies lincRNA HOTAIR probe to LSD1 albumen
Pulldown effect.Swimming lane 1 is 10%input group；Swimming lane 2 is LacZ group, uses LacZ rna probe as negative control；Swimming
Road 3 is pulldown group, uses experimental group rna probe.Figure 1A is the inventive method pulldown design sketch；Figure 1B is according to right
Method pulldown design sketch described in ratio 1；Fig. 1 C is for according to method pulldown design sketch described in comparative example 2.Send out for visible
Bright have higher protein enrichment efficiency relative to comparative example 1 method, relative to the Western of the method described in comparative example 2
The band conditions of streaking of Blot is less, and band is brighter, illustrates that this test kit protein enrichment is in hgher efficiency, highly sensitive.
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. a RNA pulldown method, it is characterised in that comprise the steps of
S1, magnetic bead pretreatment: use BSA and random oligonucleotide primer to close the magnetic bead of marked by streptavidin；
S2, prepare probe-bead complexes: be combined with biotin labeled rna probe by the magnetic bead of step S1 pretreatment；
S3, extraction pretreatment cell whole protein: in the cell whole protein extracted, add deoxyribonuclease hatch, then add
Enter unpretreated magnetic bead to hatch, remove magnetic bead, collect supernatant；
S4, pulldown: join in probe-bead complexes prepared by step S2 by the supernatant that step S3 is collected, add de-
Oxygen ribonuclease inhibitor and ribonuclease inhibitor are hatched, and collect magnetic bead, with the NT2 of qiagen rnase enzyme inhibitor
Buffer washs magnetic bead, obtains protein-probe-bead complexes；
The collection of S5, RNP: protein-probe-bead complexes step S4 prepared adds Urea CHAPS buffer and hatches,
Remove magnetic bead, collect supernatant and i.e. obtain RNP.
RNA pulldown method the most according to claim 1, it is characterised in that the side of described step S1 magnetic bead pretreatment
Method is: wash the magnetic bead of 80 μ L marked by streptavidin with 1mL 1 × TES, removes TES cleaning mixture, then adds 1mL in magnetic bead
Containing BSA and 1 × TES of random oligonucleotide primer, after incubated at room 30min, remove TES solution.
RNA pulldown method the most according to claim 2, it is characterised in that in described TES, the concentration of BSA is
0.05-0.5wt%, the concentration of random oligonucleotide primer is 10-20 μm ol/L.
RNA pulldown method the most according to claim 1 and 2, it is characterised in that oligonucleotide in described step S1
Random primer is the DNA sequence within 20bp.
RNA pulldown method the most according to claim 1, it is characterised in that described step S2 prepares probe-magnetic bead
The method of complex is: at 90 DEG C, the solution containing 2-5 μ g biotin labeling rna probe is placed 2min, is immediately placed on ice
2min, adds 50 μ LRNA structure buffer and 20 μ L DEPC water, and room temperature is placed 20min and prepared secondary structure
Rna probe；Being added in the magnetic bead of pretreatment, and add 100 μ L 2 × TES, 25 DEG C of gentle rotations mix 30-60min,
Remove supernatant；Wash magnetic bead twice with 1 × TES, remove TES.
RNA pulldown method the most according to claim 1, it is characterised in that in described step S3, pretreatment cell is complete
The method of albumen is: to 1.5 × 107The cell whole protein extract that individual cell extraction obtains adds 10 μ L DNase and 3.2 μ
L DNase Buffer, room temperature gentleness hatches 1h；Add the 40 unpretreated magnetic beads of μ L, 4 DEG C of gentle rotation 30min；Collect
Supernatant is the cell whole protein of pretreatment.
RNA pulldown method the most according to claim 1, it is characterised in that described step S3 extracts cell whole protein
Method be: with PBS wash 1.5 × 107Individual cell is once；Add 975 μ LRIP Buffer, 10 μ L PMSF solution, 10 μ L
Protease inhibitor cocktail and 5 μ L DTT solution, be homogenized 15-20 time；4 DEG C, 1500g be centrifuged 15min, collect
Supernatant is cell whole protein extract.
RNA pulldown method the most according to claim 1, it is characterised in that described step S4pulldown concrete
Method is: cell whole protein step S3 prepared joins in probe-bead complexes, and adds 472.5 μ L NT2
Buffer, 5 μ L RNase inhibitor, 15 μ g poly (dI dC), 5 μ L 0.5M EDTA solution and 2.5 μ L 0.5M
EGTA solution；Room temperature gentleness rotates hatches 2h, collects magnetic bead；Add 974 μ L NT2 buffer, 10 μ L PMSF solution, 10 μ L
Protease inhibitor cocktail, 5 μ L DTT solution and 1 μ L RNase inhibitor, wash magnetic bead, repeat
Wash four times.
RNA pulldown method the most according to claim 1, it is characterised in that the collection side of RNP in described step S5
Method is: adds 40 μ L Urea CHAPS buffer in protein-probe-bead complexes, hatches 5-10h for 4 DEG C, collect supernatant
It is probe-albumen composition.
10. a RNA pulldown test kit, it is characterised in that comprise following reagent:
Magnetic bead and reagent treatment thereof: 2 × TES solution, the magnetic bead of marked by streptavidin, random oligonucleotide primer；Described widow
The concentration of nucleotide random primer is 10-20 μm ol/L；
RNA reagent treatment: DNase, DNase Buffer, RNase inhibitor；
Protein extraction and reagent treatment: RIP buffer, Urea CHAPS buffer, protease inhibitor
Cocktail, DTT solution, PMSF solution；The concentration of described DTT solution is 100mM, and the concentration of described PMSF solution is 10mg/
mL；
Probe and combine reagent treatment: RNA structure buffer, NT2 buffer, poly (dI dC).
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Cited By (5)
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| CN106754891A (en) * | 2017-02-10 | 2017-05-31 | 广州伯信生物科技有限公司 | A kind of RNA antisenses purification process |
| CN110981929A (en) * | 2019-11-28 | 2020-04-10 | 上海市第一妇婴保健院 | For RNAm1A modified binding protein capture probe and method |
| CN111366731A (en) * | 2018-12-25 | 2020-07-03 | 中山大学孙逸仙纪念医院 | Method for detecting interaction between protein and RNA |
| CN113549676A (en) * | 2021-08-24 | 2021-10-26 | 重庆范德瓦尔斯生物科技有限公司 | Novel RNA pull down method based on magnetic beads |
| CN113817724A (en) * | 2021-09-30 | 2021-12-21 | 广州辉骏生物科技股份有限公司 | FII-RNA pulldown kit, method and application thereof |
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| CN113817724A (en) * | 2021-09-30 | 2021-12-21 | 广州辉骏生物科技股份有限公司 | FII-RNA pulldown kit, method and application thereof |
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