CN103232998A - Kit for separating RNA (ribonucleic acid) bound in RNA binding protein - Google Patents
Kit for separating RNA (ribonucleic acid) bound in RNA binding protein Download PDFInfo
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- CN103232998A CN103232998A CN2013101381275A CN201310138127A CN103232998A CN 103232998 A CN103232998 A CN 103232998A CN 2013101381275 A CN2013101381275 A CN 2013101381275A CN 201310138127 A CN201310138127 A CN 201310138127A CN 103232998 A CN103232998 A CN 103232998A
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Abstract
The invention relates to the field of molecular biology, particularly a kit for separating RNA (ribonucleic acid) bound in an RNA binding protein. The kit provided by the invention is used for separating, extracting and purifying RNA bound in a specific RNA binding protein. The kit comprises a lysis solution, a buffer solution and magnetic beads. The reagents in the kit are proportionally improved and optimized, and thus, can simply and stably obtain the mRNA (messenger ribonucleic acid) bound in the specific RNA binding protein in cells at higher yield through antibody antigen reaction and magnetic field principle. The kit is used for further cDNA real-time quantification, micro-array chip detection and other subsequent researches.
Description
Technical field
The present invention relates to biology field, relate in particular to the test kit of the RNA of a kind of separation and combination on rna binding protein.
Background technology
The regulation and control of the genetic expression aspects such as growth, differentiation and stress reaction of cell in vivo all play an important role.But except by transcription factor transcription being regulated and control, the post-transcriptional control of cell also plays an important role in physiological process, comprising (RNA-binding proteins RBPs) carries out specificity and regulation and control fast to the mRNA transcription speed by rna binding protein.At present, thisly just just begin by RBPs and the non-coding RNA Study of Mechanism to mRNA.RNA co-immunoprecipitation (RNA immunoprecipitating, RIP) be a kind of based on antigen antibody reaction, by in the method research body of co-immunoprecipitation with rna binding protein (RNAbindingprotein, RBPs) technology of the rna transcription of specificity combination relation between this.On the one hand most RNA all need and specific rna binding protein in conjunction with could stable existence in cell, and then the effect of performance translation masterplate; On the other hand, also some rna binding protein can make the RNA degraded of combination accelerate to reduce the translation skill of its corresponding protein, prevents the overexpression of some protein.This rna binding protein is most important in physiological process to the poising action of protein translation.In addition, this regulating effect the transcribing of RNA, modify, transport, all play an important role in degraded and the translation process.RIP is the antibody mediated immunity precipitated rna-albumen composition that utilizes rna binding protein, separate from the RNA-albumen composition of precipitation that obtain can be in conjunction with those RNA of specific rna binding protein, these RNA that obtain can carry out next step bioanalysis by RNA electrophoresis, RNA order-checking, quantitative RT-polymerase chain reaction (RT-PCR), gene chip analysis (RIP chip) equimolecular biological method again.Therefore, all be indispensable research method by the rna binding protein of RNA of rna binding protein isolation identification correspondence and the research of RNA itself.
But, the present method difference of using, influence factor is many, very easily contaminated samples simultaneously RNA very easily be subjected to the influence of the RNA enzyme that extensively exists again and usually cause the extremely low and RNA quality of final gained yield not guarantee.
Summary of the invention
The test kit that the purpose of this invention is to provide the mRNA of being combined with specific rna binding protein in a kind of separation and Extraction cell.
Provided by the invention for separating of the separation and Extraction test kit that is incorporated into RNA on the specific rna binding protein, comprise gentle lysate, damping fluid and magnetic bead.
Gentle lysate in the test kit comprises: concentration is the KCl of 100mM, concentration is the MgCl2 of 5mM, concentration is the 4-hydroxyethyl piperazine ethanesulfonic acid of 10mM, concentration is 0.5% tensio-active agent, and concentration is the dithiothreitol (DTT) of 100mM, and concentration is the RNA enzyme inhibitors of 100 units/mL, concentration is the phenylmethylsulfonyl fluoride of 0.05mg/mL, the Trypsin inhibitor,Trasylol of 1 μ g/mL, the leupeptin of 1 μ g/mL, the pepstatin of 1 μ g/mL.Ultrapure water after diethylpyrocarbonate is handled.
Damping fluid in the test kit comprises, pH7.4, and concentration is that the Tris of 50mM, NaCl, the concentration that concentration is 150mM are the MgCl of 1mM
2With concentration be 0.05% tensio-active agent Nonidet P-40.
It is as follows to use test kit of the present invention to carry out on the rna binding protein RNA separation steps:
1. the preparation of the mRNP lysate of cell sample: cell sample, prepare 3 * 10 for every group
7Individual cell (about 100mm culture dish 3-4), softly wash once with the perfect medium that contains foetal calf serum, wash once with precooling 10mL phosphate buffered saline buffer again, add behind an amount of 3mL phosphate buffered saline buffer that to collect the back with cell scraper centrifugal, resuspended with 4 times of precooling lysates to cell centrifugation agglomerate volume, soft resuspended back with place abundant cracking in 10 minutes on ice, the collecting cell sample is beneficial to the fragmentation of cell in-80 ℃ of store overnight.
2. second day, with cell pyrolysis liquid in thawing on ice, lysate in 4 ℃ centrifugal 30 minutes, 14000 * g, supernatant liquor are transferred in the no enzyme EP pipe, place standby on ice.
3. measure protein concentration with Bradford or Xylene Brilliant Cyanine G method, protein concentration is about 15 μ g/ μ L, the minimum lysate that needs 3mg of each sample.
4. the washing of magnetic bead is prepared, shift to an earlier date daystart: obtain even resuspended solution with the resuspended magnetic bead A of damping fluid and magnetic bead G, the 1mL magnetic bead is transferred in the EP pipe, the EP pipe is placed on the magnetic force frame, after 1 minute, inhale gently and remove supernatant, the EP pipe is taken off from the magnetic force frame, adding 2mL damping fluid gentleness is resuspended, after operating 2 times more than repeating, the EP pipe is placed on the magnetic force frame, 1 minute, inhale gently and remove supernatant, the EP pipe is taken off from the magnetic force frame, it is resuspended to add 400 μ L solution gentlenesses, divides to install to 2 EP pipes, carries out next step at once.
5. the bag quilt of antibody: antibody 200 μ g add 100 μ L magnetic bead A, IgG antibody 90 μ g add 100 μ L magnetic bead G, supply volume 1200 μ L by solution separately, place gentle suspendible on the gyroscope, 4 ℃ of bags are spent the night, with 1.6mL damping fluid washing 4 times, step is used after the washing at once with 4 respectively for magnetic bead A behind the bag quilt and magnetic bead G.
6. application of sample is as follows: bag quilt back magnetic bead 100 μ L, and DTT20 μ L, RNase OUT12 μ L, 0.5M EDTA66 μ L, cell lysate 6mg supplies volume 1.2mL at last.
7.mRNP immunoprecipitation step: behind the mixing, the gentle suspendible of room temperature 4 hours places collecting precipitation on the magnetic force frame, with 1.6mL precooling damping fluid washing 5 times, removes damping fluid at last and carries out next step at once fast.
8.mRNA extraction: each sample adds 1mL Trizol, and room temperature was placed 8 minutes, put upside down several times, the EP pipe is placed magnetic force frame last 1 minute, supernatant discarded, every pipe adds the 0.2mL chloroform, vortex, room temperature was placed 2-3 minute, and 4 ℃ of centrifugal 5 minutes 12000 * g collect supernatant (about 0.5-0.6mL), the Virahol that adds same volume, 5 μ L glycogen put upside down vortex, hatch 5-10 minute for-20 ℃, 4 ℃, centrifugal 10 minutes of 12000 * g abandons supernatant, washs with 75% ethanol vortex, 4 ℃, centrifugal 10 minutes of 12000 * g, heavy molten with 30 μ L nuclease free water after volatilizing, obtain RNA.
The general reagent that needs to prepare in addition comprises: Trizol, chloroform, ethanol, Virahol, phosphate buffered saline buffer.
The present invention is by the mRNA that is specific RBPs specific combination that above step obtains, and can be further purified evaluation, quantitative analysis or be used for research such as micro-array chip.The equal ratio of each reagent is through improving and optimizating in this test kit, can be easy, stable, obtain the mRNA of being combined with specific rna binding protein in the cell with higher yields, and for follow-up studies such as the real-time quantitative of further cDNA and micro-array chip detections.
Description of drawings
Fig. 1 represents that fluorescent quantitative PCR technique detects the expression level of the protein bound COX-2 of HuR in normal group and model group.
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is further specified, but the present invention far is not limited only to embodiment.
Embodiment 1
To LPS stimulate rna binding protein HuR in the Raw264.7 cell of back in conjunction with the separation of mRNA
1, reagent source:
(1) magnetic bead A is the Dynabeads of Invitrogen company;
(2) magnetic bead G is the Dynabeads of Invitrogen company;
(3) mouse IgG of the present invention produces for Sigma company;
(4) the RNA enzyme inhibitors is for producing Rnase OUT for Invitrogen company;
(5) proteolytic enzyme mixed inhibitor Roche company produces;
(6) KCl, MgCl
2, Hepes, tensio-active agent Nonidet P-40, dithiothreitol (DTT), 4-hydroxyethyl piperazine ethanesulfonic acid are Sigma company and produce.
2, instrument source:
(1) refrigerated centrifuge, Sigma;
(2) microplate reader, iQ5Multi color Red-time PCR Deteetion System is Bio-Rad;
(3) four-dimensional gyroscope, its woods Bel;
(4) magnetic force frame, Invitrogen;
(5) Gene Quent nucleic acid quantification instrument, GE company;
1. the preparation of the mRNP lysate of cell sample: the Raw264.7 cell, add LPS (50ng/mL), stimulated 12 hours, establish the contrast of blank and LPS model, every group of sample prepares 3 * 10
7Individual cell (about 100mm culture dish 3-4), softly wash once with the perfect medium that contains FBS, wash once with precooling 10mL PBS damping fluid again, add behind an amount of 3mL PBS that to collect the back with cell scraper centrifugal, resuspended with 4 times of precooling lysates to cell centrifugation agglomerate volume, soft resuspended back with place abundant cracking in 10 minutes on ice, the collecting cell sample is beneficial to the fragmentation of cell in-80 ℃ of store overnight.
2. second day, with cell pyrolysis liquid in thawing on ice, lysate in 4 ℃ centrifugal 30 minutes, 14000 * g, supernatant liquor are transferred in the no enzyme EP pipe, place standby on ice.
3. measure protein concentration with Bradford or Xylene Brilliant Cyanine G method, protein concentration is about 15 μ g/ μ L, the minimum lysate that needs 3mg of each sample.
4. the washing of magnetic bead is prepared, shift to an earlier date daystart: obtain even resuspended solution with the resuspended magnetic bead A of damping fluid and magnetic bead G, the 1mL magnetic bead is transferred in the EP pipe, the EP pipe is placed on the magnetic force frame, after 1 minute, inhale gently and remove supernatant, the EP pipe is taken off from the magnetic force frame, adding 2mL damping fluid gentleness is resuspended, after operating 2 times more than repeating, the EP pipe is placed on the magnetic force frame, 1 minute, inhale gently and remove supernatant, the EP pipe is taken off from the magnetic force frame, it is resuspended to add 400 μ L solution gentlenesses, divides to install to 2 EP pipes, carries out next step at once.
5. the bag quilt of antibody: HuR antibody 400 μ g add 200 μ L magnetic bead A, IgG antibody 180 μ g add 200 μ L magnetic bead G, supply volume 1200 μ L by solution separately, place gentle suspendible on the gyroscope, 4 ℃ of bags are spent the night, with 1.6mL damping fluid washing 4 times, step is used after the washing at once with 4 respectively for magnetic bead A behind the bag quilt and magnetic bead G.
6. application of sample is as follows: bag quilt back magnetic bead 100 μ L, and DTT20 μ L, RNase OUT12 μ L, 0.5MEDTA66 μ L, cell lysate 6mg supplies volume 1.2mL at last.
7.mRNP immunoprecipitation step: behind the mixing, the gentle suspendible of room temperature 4 hours places collecting precipitation on the magnetic force frame, with 1.6mL precooling damping fluid washing 5 times, removes damping fluid at last and carries out next step at once fast.
8.mRNA extraction: each sample adds 1mL Trizol, and room temperature was placed 8 minutes, put upside down several times, the EP pipe is placed magnetic force frame last 1 minute, supernatant discarded, every pipe adds the 0.2mL chloroform, vortex, room temperature was placed 2-3 minute, 4 ℃ of centrifugal 5 minutes 12000 * g, collect supernatant (about 0.5-0.6mL), the Virahol that adds same volume, 5 μ L glycogen put upside down vortex, hatched 5-10 minute for-20 ℃, 4 ℃, centrifugal 10 minutes of 12000 * g abandons supernatant, wash with 75% ethanol vortex, 4 ℃, centrifugal 10 minutes of 12000 * g volatilizes the back and weighs molten with 30 μ L nuclease free water, detect by the nucleic acid quantification instrument, it is as follows to obtain quantitative result:
Table 1RNA is in conjunction with protein immunization co-precipitation gained RNA concentration
9. reverse transcription:
Following component is added in the pipe successively, uses Takara reverse transcription test kit to be example:
Table 2 reverse transcription system application of sample component and application of sample amount
Flick the gentle mixing of tube wall, 37 ℃ of incubation 15min, 85 ℃ 5 seconds, 4 ℃ 2 minutes, carry out next step.
10. the detection of fluorescent quantitation: get 0.2ml PCR eight connecting legs, add following component successively, use the Evagreen reagent of Bio-rad to be example: (cumulative volume 20 μ L)
Table 3 fluorescent quantitation detection architecture application of sample component and application of sample amount
The vibration mixing, of short duration centrifugal, react by following cycling condition:
After the condition of real-time quantitative PCR amplified reaction is 95 ℃ of pre-sex change, 1 circulation in 3 minutes, 95 ℃ of each circulation sex change 10 seconds, anneal 60 ℃ 10 seconds, after totally 40 circulations, extend 65 ℃ 10 seconds, the fluorescent signal after the cycle annealing is gathered in totally 61 circulations.Be confidential reference items with β-actin during interpretation of result, the destination gene expression that detects COX-2 expression levels normal control group is 1, the results are shown in Figure 1.The primer sequence is seen the following form by Literature Consult.
The mouse primer sequence of having reported that table 4 present embodiment is used
Claims (4)
1. one kind for separating of the separation and Extraction test kit that is incorporated into RNA on the specific rna binding protein, it is characterized in that, comprises lysate, damping fluid and magnetic bead.
2. separation and Extraction test kit according to claim 1 is characterized in that, described lysate comprises: concentration is the KCl of 100mM, and concentration is the MgCl of 5mM
2Concentration is the 4-hydroxyethyl piperazine ethanesulfonic acid of 10mM, concentration is 0.5% tensio-active agent, concentration is the dithiothreitol (DTT) of 100mM, concentration is the RNA enzyme inhibitors of 100 units/mL, and concentration is the phenylmethylsulfonyl fluoride of 0.05mg/mL, the Trypsin inhibitor,Trasylol of 1 μ g/mL, the leupeptin of 1 μ g/mL, the pepstatin of 1 μ g/mL.
3. separation and Extraction test kit according to claim 2 is characterized in that, described lysate prescription liquid medium is the ultrapure water after diethylpyrocarbonate is handled.
4. separation and Extraction test kit according to claim 1 is characterized in that, described damping fluid comprises, pH7.4, and concentration is that the Tris of 50mM, NaCl, the concentration that concentration is 150mM are the MgCl of 1mM
2With concentration be 0.05% tensio-active agent.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106191036A (en) * | 2016-08-08 | 2016-12-07 | 吴江近岸蛋白质科技有限公司 | Rna binding protein is at the application extracted in nucleic acid and extracting method |
| CN108427000A (en) * | 2017-02-15 | 2018-08-21 | 广州市锐博生物科技有限公司 | A kind of method and kit of capture nucleic acid binding protein |
| CN111269964A (en) * | 2020-02-20 | 2020-06-12 | 上海纽仁生物医药科技有限公司 | Kit for RNA immunoprecipitation by using protein antibody |
| CN111458518A (en) * | 2020-04-03 | 2020-07-28 | 中国医科大学 | Development and application of simple RNA binding protein immunoprecipitation kit |
| CN111826419A (en) * | 2020-07-06 | 2020-10-27 | 重庆生命知源科技有限公司 | Library-establishing sequencing method suitable for RIP (RIP-induced plasticity) experiment of trace cells |
-
2013
- 2013-04-22 CN CN2013101381275A patent/CN103232998A/en active Pending
Non-Patent Citations (3)
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| MERTENS ET AL: "Nuclear particles containing RNA polymerase III complexes associated with the junctional plaque protein plakophilin 2", 《PNAS》 * |
| 崔亚丽等: "磁性微粒在核酸研究中的应用", 《西北农业大学学报》 * |
| 符向辉: "XR1P蛋白结合结构域的鉴定及相关小分子RNA的筛选", 《中国优秀硕士论文全文数据库 医药卫生科技辑》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106191036A (en) * | 2016-08-08 | 2016-12-07 | 吴江近岸蛋白质科技有限公司 | Rna binding protein is at the application extracted in nucleic acid and extracting method |
| CN108427000A (en) * | 2017-02-15 | 2018-08-21 | 广州市锐博生物科技有限公司 | A kind of method and kit of capture nucleic acid binding protein |
| CN108427000B (en) * | 2017-02-15 | 2021-06-08 | 广州市锐博生物科技有限公司 | Method and kit for capturing nucleic acid binding protein |
| CN111269964A (en) * | 2020-02-20 | 2020-06-12 | 上海纽仁生物医药科技有限公司 | Kit for RNA immunoprecipitation by using protein antibody |
| CN111269964B (en) * | 2020-02-20 | 2021-03-02 | 上海纽仁生物医药科技有限公司 | Kit for RNA immunoprecipitation by using protein antibody |
| CN111458518A (en) * | 2020-04-03 | 2020-07-28 | 中国医科大学 | Development and application of simple RNA binding protein immunoprecipitation kit |
| CN111458518B (en) * | 2020-04-03 | 2023-10-27 | 中国医科大学 | Development and application of simple RNA binding protein immunoprecipitation kit |
| CN111826419A (en) * | 2020-07-06 | 2020-10-27 | 重庆生命知源科技有限公司 | Library-establishing sequencing method suitable for RIP (RIP-induced plasticity) experiment of trace cells |
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Application publication date: 20130807 |