CN103966317A - Ultrasound breaking method for animal tissue in chromatin co-immunoprecipitation - Google Patents

Abstract

The invention provides an ultrasonic breaking method for animal tissue in chromatin co-immunoprecipitation. The ultrasound breaking method comprises the steps as follows: firstly, adding a mixture of a collagenase solution and a DPBS solution at a proportion of 1:1 into animal tissue for an experiment; shaking for digesting the animal tissue at the temperature of 37 DEG C; centrifugalizing; removing supernatant fluid; performing conventional ultrasound breaking. The ultrasound breaking method can better acquire a DNA-protein compound segment with a proper size, and a foundation is laid for the success of the whole ChIP experiment.

Description

The ultrasonication method of animal tissues in chromatin co-immunoprecipitation

Technical field

The present invention relates to immunoprecipitation field, specifically, relate to the ultrasonication method of animal tissues in a kind of chromatin co-immunoprecipitation.

Background technology

Biological gene regulating and to express be extremely complicated but orderly process, and organism genomic dna exists with chromatin form in cell, and protein and DNA to do be mutually the important foundation of cell functionating.Therefore, genetic expression and regulation and control model thereof can be further understood in Study on Protein and the DNA interaction under chromatin environment.

At present, the common methods of researching DNA-protein interaction have (KM Sullivan such as Westernblotting, electrophoretic mobility shift assay, footprinting, R Storb, CDBuckner, et al.Graft-versus-host disease as adoptive immunotherapy inpatients with advanced hematologic neoplasms.N.Engl.J.Med., 1989,320:828 – 834), but all there is the limitation that can not truly reflect that under physiological condition, DNA-protein is done mutually in these methods.Compare with these conventional arts, chromatin Immunoprecipitation (Chromatin Immunoprecipitation, ChIP) be researching DNA-protein interactional standard method in vivo (Rusk N.Reverse ChIP.NatMethods, 2009,6 (3): 187).Chromatin Immunoprecipitation can positioning analysis body internal protein and the action site of DNA, use ChIP technology to combine with additive method, for example combine and can obtain the single transcription factor distribution collection of illustrative plates of whole genomic level, trans factor Binding in vivo site with the technology such as footprinting in superchip (microarray), order-checking (sequencing), body, and system discloses the genetic mechanism of the epigenetics such as nucleosome location, histone modification.

The general technology flow process of ChIP technology is: mixture → cleanings of antibody-protein-dna, the wash-out of formaldehyde fixed cell, cross-linked proteins and chromatin ultrasonication cross-linked composite → collection ultrasonication product → add respectively object, cloudy be combined with target DNA-albumen composition → magnetic bead of reference protein antibody absorptive collection precipitation precipitate the mixture → solution obtaining and be cross-linked, and DNA fragmentation → PCR that purifying obtains detects precipitation result.

Successfully chromatin immunoprecipitation is the prerequisite of the follow-up high-flux sequence of experiment or chip hybridization, is the prerequisite of whole chromatin co-immunoprecipitation Success in Experiment but utilize suitable ultrasound condition to obtain the suitable DNA-chromatin mixture of clip size.At present, all for directly utilizing, cell is cross-linked traditional experimental program, broken and precipitation, but use traditional method, to utilizing, animal tissues is directly cross-linked, broken chromatin co-immunoprecipitation experiment is difficult to obtain desirable result.

Summary of the invention

In order to solve problems of the prior art, the object of this invention is to provide the ultrasonication method of animal tissues in a kind of chromatin co-immunoprecipitation, the DNA-protein that it is suitable that the method can obtain clip size is beneficial to subsequent experimental step.

In order to realize the object of the invention, the invention provides the ultrasonication method of animal tissues in a kind of chromatin co-immunoprecipitation, described ultrasonication method first by animal tissues through collagenase digesting, be specially: add collagenase mixing solutions to animal for research tissue, 37 DEG C of concussions digest animal tissues until animal tissues's piece dissolves completely, 4 DEG C, after 700g-900g is centrifugal, remove supernatant;

The addition of described collagenase mixing solutions is that every 0.01 gram of animal tissues adds 150-250ul mixing solutions;

Described collagenase mixing solutions is collagenase solution: the solution of DPBS solution (Gibco, C14190, the U.S.)=1:1.

Described collagenase solution is that collagenase (I type) (Sigma, C0130, the U.S.) 100mg and BSA (serum albumin) 2g are dissolved in PBS100ml, mixes to be made into.

As preferably, the time of described concussion digestion is 1.5-3.5h.

Further, described ultrasonication method also comprises the steps:

1) animal tissues is through collagenase digesting, goes after supernatant after centrifugal, and what add that 500-1000ul contains proteinase inhibitor organizes stationary liquid TSS/PI, the centrifugal supernatant that goes;

2) adding formaldehyde final concentration is 1 × phosphate buffered saline buffer (PBS) mixing solutions of 1%, and under room temperature, hatching is crosslinked;

3) add 10 × glycine solution, room temperature hatching, removes supernatant after centrifugal;

4) add the 1 × PBS solution that contains proteinase inhibitor, hatch on ice, remove supernatant after centrifugal, repeat twice;

5) add the cytolysis damping fluid TLB/PI that contains proteinase inhibitor, hatch centrifugal going after supernatant on ice;

6) add the ChIP diluent that contains proteinase inhibitor;

7) ultrasonication DNA-protein complex, collects the product that different ultrasound conditions obtain, and adds RNaseA, hatches 30min for 37 DEG C;

8) add Proteinase K, hatch 2h for 62 DEG C;

9) agarose electrophoresis screening clip size concentrates on the ultrasonication product of 200-500bp.

Clip size concentrates on the ultrasonication product of 200-500bp, carry out subsequent experimental step: add respectively mixture → cleanings of antibody-protein-dna, the wash-out of object, cloudy be combined with target DNA-albumen composition → magnetic bead of reference protein antibody absorptive collection precipitation to precipitate the mixture → solution obtaining and be cross-linked, DNA fragmentation → PCR that purifying obtains detects precipitation result.

Beneficial effect of the present invention is:

The present invention has overcome traditional C hIP method and has not been suitable for the shortcoming of animal tissues, before carrying out crosslinked, the ultrasonication step of ChIP experiment to the method that adds collagenase digesting in animal tissues, can better obtain the DNA-protein complex that clip size is suitable, for the success of whole ChIP experiment is laid a good foundation.

The primary cell digestion that collagenase is mainly used in cell cultures is in the prior art cultivated, and general application target carries out cell cultures for digesting primary cell, and the present invention uses collagenase digesting tissue for chromatin co-immunoprecipitation.

Use this method can reduce the number of times of groping to animal tissues's piece ultrasonication condition, not only saved the consumption to laboratory sample, and saved experiment and used reagent consumptive material, comprise your reagent such as ChIP process test kit.Meanwhile, also can reduce time consumption and the energy consumption of experimentation to experimenter.

Brief description of the drawings

Fig. 1 is in the embodiment of the present invention 1 under identical ultrasound condition, adds collagenase digesting and the animal tissues's sample ultrasonic result comparison that does not add collagenase digesting;

Wherein: A. carries out ultrasonication sample after adding collagenase digesting; B. do not add collagenase digesting directly to carry out ultrasonication sample; 1. sample before supersound process; 2. supersound process 10min; 3. supersound process 20min; 4. supersound process 30min; 5. supersound process 40min; M:Marker.

Fig. 2 is under the condition of subsequent experimental process identical in the embodiment of the present invention 1, whether adds the comparison of collagen protein enzymic digestion animal tissues on whole ChIP experiment impact;

Wherein: after A. adds collagenase digesting, ultrasonic sample ChIP result PCR checks; B. do not add the direct ultrasonic sample ChIP result PCR inspection of collagen protein enzymic digestion; 1. the object antibody mediated immunity precipitation DNA(positive); 2.Igg antibody mediated immunity precipitation DNA(feminine gender); 3.Input DNA; 4.DNA contrast; M:Marker.

Fig. 3 is in the embodiment of the present invention 2 under identical ultrasound condition, adds collagenase digesting and the animal tissues's sample ultrasonic result comparison that does not add collagenase digesting;

Wherein: A. carries out ultrasonication sample after adding collagenase digesting; B. do not add collagenase digesting directly to carry out ultrasonication sample; 1. sample before supersound process; 2. supersound process 10min; 3. supersound process 20min; 4. supersound process 30min; 5. supersound process 40min; M:Marker.

Embodiment

Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.

The ultrasonication method (taking chicken embryo as example) of animal tissues in embodiment 1 chromatin co-immunoprecipitation

One, ChIP laboratory sample preparation

Collect after the sterilization of kind of egg, be placed in 37.8 DEG C of temperature, humidity 60%, in every 2 hours egg-turnings brooder once, hatching, to the 16th day, gathers chicken embryo heart tissue, and is frozen in rapidly in liquid nitrogen.

Two, in ChIP experiment flow, ultrasonication experiment condition is explored and result verification

In experiment, agents useful for same is except indicating especially, from ChIP ^tMg Tissue Kit(Millipore, the U.S.)

Get the about 50ug of freezing Embryo Gallus domesticus heart tissue, slowly thaw on ice and shred → add the Collagenase (Sigma that 1ml1:1 mixes, C0130, the U.S.), DPBS (Gibco, C14190, the U.S.) solution, 37 DEG C of shaking water bath digestion animal tissues 2.5h → 4 DEG C, the centrifugal 15min of 800g, remove supernatant → add 500ul TSS/PI, with rifle head repeatedly blow and beat → 4 DEG C, the centrifugal 5min of 800g, go supernatant → add 1ml1 × phosphate buffered saline buffer (PBS)/1% formaldehyde solution, the crosslinked 10min of hatching under room temperature → add 100ul10 × glycine solution, room temperature hatching 5min → 4 DEG C, the centrifugal 5min of 800g, remove supernatant → add 1ml1 × PBS/PI, hatch 5min → 4 DEG C on ice, after the centrifugal 5min of 800g, remove supernatant, repeat twice → 4 DEG C of previous steps, after the centrifugal 5min of 800g, remove supernatant, add 500ul TLB/PI, hatch 20min on ice, once → 4 DEG C of every 5min vortexs, after the centrifugal 5min of 800g, remove supernatant, add the each 10ul of product that the different ultrasound conditions of ChIP diluent 500ul → ultrasonication DNA-protein complex of containing proteinase inhibitor (concrete ultrasound condition is as follows) → collect obtain → add glad remittances of 1ul RNaseA(sky, China), hatch 30min → add 0.8ul Proteinase K (glad remittance sky for 37 DEG C, China), hatch 2h → 1.2% agarose gel for 62 DEG C, 120V voltage electrophoresis 30min, detect ultrasonication product and whether meet subsequent experimental requirement.

Ultrasonication condition: use Ningbo new sesame TZ-IID Ultrasonic Cell Disruptor, power 50%(450W), intermittently 50s of work 10s, every 10min samples once for detection of judging the most suitable working hour.Identical ultrasound condition, adds collagenase digesting and the animal tissues's sample ultrasonic result that does not add collagenase digesting relatively to see Fig. 1.As seen from Figure 1, under identical ultrasound condition, add the sample of collagenase digesting can better reach subsequent experimental requirement, the clip size of DNA-protein complex concentrates between 200-500bp, and there is no genome master tape; And do not add the sample of collagenase digesting under identical ultrasonic number of times, still exist the clip size of genome master tape and mixture there is no concentrated area.In conjunction with our early stages a large amount of Experimental Ultrasonic conditions grope, while finding not add animal tissues's sample of collagenase digesting directly to carry out ultrasonication, be to strengthen ultrasonic power or extend ultrasonic time all to reach subsequent experimental requirement.Therefore, present method has effectively overcome traditional C hIP method and has not been suitable for the shortcoming of animal tissues.

For checking the impact of two kinds of processing on last chromatin co-immunoprecipitation experimental result, above ultrasonic sample is carried out to ChIP subsequent experimental step: add respectively mixture → cleanings of antibody-protein-dna, the wash-out of object, be combined with target DNA-albumen composition → magnetic bead of cloudy reference protein antibody (Igg) absorptive collection precipitation to precipitate the mixture → solution obtaining and be cross-linked, DNA fragmentation → PCR that purifying obtains detects precipitation result.Subsequent experimental process adopts ChIP ^tMgTissue Kit(Millipore, the U.S.) carry out.Fig. 2 is identical subsequent experimental process, and whether contrast adds collagenase digesting animal tissues on whole ChIP Success in Experiment whether impact.

In object antibody mediated immunity precipitation DNA, Igg antibody mediated immunity precipitation DNA, Input DNA and 4 parts of DNA samples of conventional animal genomic dna, respectively specific target gene and crt gene (housekeeping gene) being carried out to pcr amplification is to detect the whether successful important Quality Control step of chromatin co-immunoprecipitation.Amplification shows (Fig. 2), adds ultrasonic animal tissues's sample after collagenase digesting, the precipitation that object antibody is special target gene, and to housekeeping gene expanding effect extremely a little less than.The sample adding after collagenase digesting is described, chromatin immunoprecipitation experiment result is successful.But, do not add the animal tissues's sample after collagenase digesting, in Igg antibody mediated immunity precipitation, also there is precipitation, illustrate in chromatin immunoprecipitation experiment and have a large amount of non-specific enrichments, experimental result can think that precipitation enrichment is failed.Therefore whether success is most important to whole ChIP experiment, whether to add collagenase digesting animal tissues sample.

The ultrasonication method (taking porcine tissue as example) of animal tissues in embodiment 2 chromatin co-immunoprecipitations

One, ChIP laboratory sample preparation

Gather 6 the monthly age pig heart tissue, and be frozen in rapidly in liquid nitrogen.

Two, in ChIP experiment flow, ultrasonication experiment condition is explored and result verification

In experiment, agents useful for same is except indicating especially, from ChIP ^tMg Tissue Kit(Millipore, the U.S.)

Get freezing pig heart and organize about 50ug, slowly thaw on ice and shred → add the Collagenase (Sigma that 1ml1:1 mixes, C0130, the U.S.), DPBS (Gibco, C14190, the U.S.) solution, 37 DEG C of shaking water bath digestion animal tissues 3.5h → 4 DEG C, the centrifugal 15min of 800g, remove supernatant → add 500ul TSS/PI, with rifle head repeatedly blow and beat → 4 DEG C, the centrifugal 5min of 800g, go supernatant → add 1ml1 × phosphate buffered saline buffer (PBS)/1% formaldehyde solution, the crosslinked 10min of hatching under room temperature → add 100ul10 × glycine solution, room temperature hatching 5min → 4 DEG C, the centrifugal 5min of 800g, remove supernatant → add 1ml1 × PBS/PI, hatch 5min → 4 DEG C on ice, after the centrifugal 5min of 800g, remove supernatant, repeat twice → 4 DEG C of previous steps, after the centrifugal 5min of 800g, remove supernatant, add 500ul TLB/PI, hatch 20min on ice, once → 4 DEG C of every 5min vortexs, after the centrifugal 5min of 800g, remove supernatant, add the each 10ul of product that the different ultrasound conditions of ChIP diluent 500ul → ultrasonication DNA-protein complex of containing proteinase inhibitor (concrete ultrasound condition is as follows) → collect obtain → add glad remittances of 1ul RNaseA(sky, China), hatch 30min → add 0.8ul Proteinase K (glad remittance sky for 37 DEG C, China), hatch 2h → 1.2% agarose gel for 62 DEG C, 120V voltage electrophoresis 30min, detect ultrasonication product and whether meet subsequent experimental requirement.

Ultrasonication condition: use Ningbo new sesame TZ-IID Ultrasonic Cell Disruptor, power 50%(450W), intermittently 50s of work 10s, every 10min samples once for detection of judging the most suitable working hour.Identical ultrasound condition, adds collagenase digesting and the animal tissues's sample ultrasonic result that does not add collagenase digesting relatively to see Fig. 3.As seen from Figure 3, under identical ultrasound condition, add the sample of collagenase digesting can better reach subsequent experimental requirement, the clip size of DNA-protein complex concentrates between 200-500bp, and there is no genome master tape; And do not add the sample of collagenase digesting under identical ultrasonic number of times, still exist the clip size of genome master tape and mixture there is no concentrated area.In conjunction with our early stages a large amount of Experimental Ultrasonic conditions grope, while finding not add animal tissues's sample of collagenase digesting directly to carry out ultrasonication, be to strengthen ultrasonic power or extend ultrasonic time all to reach subsequent experimental requirement.Therefore, present method has effectively overcome traditional C hIP method and has not been suitable for the shortcoming of animal tissues.

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims (4)

1. the ultrasonication method of animal tissues in a chromatin co-immunoprecipitation, it is characterized in that, described ultrasonication method first by animal tissues through collagenase digesting, be specially: add collagenase mixing solutions to animal for research tissue, 37 DEG C of concussions digest animal tissues until animal tissues's piece dissolves completely, 4 DEG C, after 700g-900g is centrifugal, remove supernatant;

The addition of described collagenase mixing solutions is that every 0.01 gram of animal tissues adds 150-250ul mixing solutions;

Described collagenase mixing solutions is collagenase solution: the solution of DPBS solution=1:1.

2. ultrasonication method according to claim 1, is characterized in that, the time of described concussion digestion is 1.5-3.5h.

3. ultrasonication method according to claim 1, is characterized in that, described collagenase solution is for to be dissolved into collagenase 100mg and serum albumin BSA2g in 100mlPBS, mixes to be made into.

4. ultrasonication method according to claim 1, is characterized in that, described ultrasonication method also comprises the steps:

1) animal tissues is through collagenase digesting, goes after supernatant after centrifugal, adds to organize stationary liquid TSS/PI, the centrifugal supernatant that goes containing proteinase inhibitor;

2) adding formaldehyde final concentration is 1 × phosphate buffered saline buffer (PBS) mixing solutions of 1%, and under room temperature, hatching is crosslinked;

3) add 10 × glycine solution, room temperature hatching, removes supernatant after centrifugal;

4) add the 1 × PBS solution that contains proteinase inhibitor, hatch on ice, remove supernatant after centrifugal, repeat twice;

5) add the cytolysis damping fluid TLB/PI that contains proteinase inhibitor, hatch centrifugal going after supernatant on ice;

6) add the ChIP diluent that contains proteinase inhibitor;

7) ultrasonication DNA-protein complex, collects the product that different ultrasound conditions obtain, and adds RNaseA, hatches 30min for 37 DEG C;

8) add Proteinase K, hatch 2h for 62 DEG C;

9) agarose electrophoresis screening clip size concentrates on the ultrasonication product of 200-500bp.